Regulation of oncogenes by microRNAs

ABSTRACT

Naturally occurring miRNAs that regulate human oncogenes and methods of use therof are described. Suitable nucleic acids for use in the methods and compositions described herein include, but are not limited to, pri-miRNA, pre-miRNA, mature miRNA or fragments of variants thereof that retain the biological activity of the mature miRNA and DNA encoding a pri-miRNA, pre-miRNA, mature miRNA, fragments or variants thereof, or regulatory elements of the miRNA. The compositions containing nucleic acids are administered to a patient in need of treatment or prophylaxis of at least one symptom or manifestation of cancer. In one embodiment, the compositions are administered in an effective amount to inhibit gene expression of one or more oncogenes. Methods for treatment or prevention of at least one symptom or manifestation of cancer are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 60/606,855 filed Sep.2, 2004 entitled “Regulation of Oncogenes by MicroRNAs” by Frank J.Slack, Steven M. Johnson, Kristy L. Reinert, and Helge Grosshans.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

The Federal Government has certain rights in this invention by virtue ofGrant No. 1R01GM062594-01A1 and Grant No. 1R01GM064701-01 from theNational Institute of Health to Frank J. Slack.

BACKGROUND OF THE INVENTION

Cancer is a group of diseases characterized by uncontrolled growth andspread of abnormal cells. Cancer is caused by both external factors(tobacco, chemicals, radiation, and infections organisms) and internalfactors (inherited mutations, hormones, immune conditions, and DNAdamage). These factors may act together or sequentially to initiateand/or promote carcinogenesis. Cancer causes 1 of every 4 deaths and isthe leading cause of death in people under age 85 in the United States.Nearly half of all men and a little over one third of all women in theU.S. will develop cancer during their lifetimes. Today, millions ofpeople are living with cancer or have had cancer. The sooner a cancer isfound and treatment begins, the better are the chances for living formany years.

Lung cancer is the leading cause of cancer deaths for both men and womenin the United States. According to the American Cancer Society, about160,000 people die annually of this disease, with about 170,000 newlydiagnosed cases each year. Despite the use of surgery, chemotherapy, andradiation, the survival rate for patients remains extremely poor (>15%over 5 years). As estimated by the American Cancer Society, the 5-yearsurvival rate for all cancers is about 64% for cancers diagnosed between1995-2000. However, the survival rate varies depending on cancer typeand the stage of cancer at time of detection. For example, the survivalrate for brain, breast, and colon cancer is 33, 88, and 63%,respectively for cancers diagnosed between 1995-2000. Therefore,treatments in addition to the standard methods of treatment that includesurgery, radiation, chemotherapy, immunotherapy, and hormone therapy areneeded.

Misregulation of genes that control cell fate determination oftencontributes to cancer. Such altered genes are known as oncogenes.Oncogenes are called proto-oncogenes when they are normal (i.e., notmutated). Proto-oncogenes encode components of the cell's normalgrowth-control pathway. Some of these components are growth factors,receptors, signaling enzymes, and transcription factors.

Ras is one such oncogene. Mammalian ras genes code for closely related,small proteins (H-ras, K-ras and N-ras). Ras is found in normal cells,where it helps to relay signals by acting as a switch. When receptors onthe cell surface are stimulated (by a hormone, for example), Ras isswitched on and transduces signals that tell the cell to grow. If thecell-surface receptor is not stimulated, Ras is not activated and so thepathway that results in cell growth is not initiated. In about 30% ofhuman cancers, Ras is mutated so that it is permanently switched on,telling the cell to grow regardless of whether receptors on the cellsurface are activated or not. A high incidence of ras gene mutations isfound in all lung cancers and adenocarcinomas.(10% and 25%,respectively, K-ras), in malignant tumors of the pancreas (80-90%,K-ras), in colorectal carcinomas (30-60%, K-ras), in non-melanoma skincancer (30-50%, H-ras), in hematopoietic neoplasia of myeloid origin(18-30%, K-and N-ras), and in seminoma (25-40%, K-ras). In other tumors,a mutant ras gene is found at a lower frequency: for example, in breastcarcinoma (0-12%, K-ras), glioblastoma and neuroblastoma (0-10%, K- andN-ras).

Other oncogenes include members of the MYC family (c-MYC, N-MYC, andL-MYC), which have been widely studied, and amplification of myc geneshas been found in a variety of tumor types including lung (c-MYC, N-MYC,L-MYC), colon (c-MYC), breast (c-MYC), and neuroblastoma (N-MYC). Genesthat inhibit apoptosis have also been identified as oncogenes. Theprototype of these genes is BCL-2. Originally identified at thechromosomal breakpoint in follicular lymphoma, this protein was found toinhibit cell death rather than promote cell growth. BCL-2 belongs to afamily of intracellular proteins whose role is to regulate caspaseactivation that leads to DNA fragmentation and cell death. In melanoma,BCL-2 has been reported to be overexpressed in primary and metastaticlesions and this phenotype is associated with tumor progression.

Micro RNAs (referred to as “miRNAs”) are small non-coding RNAs,belonging to a class of regulatory molecules found in plants and animalsthat control gene expression by binding to complementary sites on targetmessenger RNA (mRNA) transcripts (FIG. 1) (SEQ ID Nos. 1, 2 and 3).miRNAs are generated from large RNA precursors (termed pri-miRNAs) thatare processed in the nucleus into approximately 70 nucleotidepre-miRNAs, which fold into imperfect stem-loop structures (Lee, Y., etal., Nature (2003) 425(6956):415-9) (FIG. 1). The pre-miRNAs undergo anadditional processing step within the cytoplasm where mature miRNAs of18-25 nucleotides in length are excised from one side of the pre-miRNAhairpin by an RNase III enzyme, Dicer (Hutvagner, G., et al., Science(2001) 12:12 and Grishok, A., et al., Cell (2001) 106(1):23-34). MiRNAshave been shown to regulate gene expression in two ways. First, miRNAsthat bind to protein-coding mRNA sequences that are exactlycomplementary to the miRNA induce the RNA-mediated interference (RNAi)pathway. Messenger RNA targets are cleaved by ribonucleases in the RISCcomplex. This mechanism of miRNA-mediated gene silencing has beenobserved mainly in plants (Hamilton, A. J. and D. C. Baulcombe, Science(1999) 286(5441):950-2 and Reinhart, B. J., et al., MicroRNAs in plants.Genes and Dev. (2002) 16:1616-1626), but an example is known fromanimals (Yekta, S., I. H. Shih, and D. P. Bartel, Science (2004)304(5670):594-6). In the second mechanism, miRNAs that bind to imperfectcomplementary sites on messenger RNA transcripts direct gene regulationat the posttranscriptional level but do not cleave their mRNA targets.MiRNAs identified in both plants and animals use this mechanism to exerttranslational control of their gene targets (Bartel, D. P., Cell (2004)116(2):281-97).

Hundreds of miRNAs have been identified in the fly, worm, plant andmammalian genomes. The biological role for the majority of the miRNAsremains unknown because almost all of these were found through cloningand bioinformatic approaches (Lagos-Quintana, M., et al., Curr Biol(2002) 12(9):735-9; Lagos-Quintana, M., et al., RNA (2003) 9(2):175-179; Lagos-Quintana, M., et al., Science (2001) 294(5543): 853-8;Lee, R. C. and V. Ambros, Science (2001) 294(5543):862-4; Lau, N. C., etal., Science (2001) 294(5543):858-62; Lim, L. P., et al., Genes Dev(2003) 17(8):991-1008; Johnston, R. J. and O. Hobert, Nature (2003)426(6968):845-9; and Chang, S., et al. Nature (2004) 430(7001):785-9).

It is likely that these uncharacterized miRNAs act as important generegulators during development to coordinate proper organ formation,embryonic patterning, and body growth, but this remains to beestablished. In zebrafish, most miRNAs are expressed from organogenesisonward (Chen, P. Y., et al., Genes Dev (2005) 19(11):1288-93 andWienholds, E., et al., Science, (2005)).

The biological roles for several miRNAs have been elucidated. Thesestudies highlight the importance of these regulatory molecules in avariety of developmental and metabolic processes. For example, theDrosophila miRNA, bantam, was identified in a gain-of-function geneticscreen for factors that caused abnormal tissue growth (Brennecke, J., etal., Cell (2003) 113(l):25-36). Bantam was found to induce tissue growthin the fly by both stimulating cell proliferation and inhibitingapoptosis (Brennecke, J., et al., Cell (2003). 113(1):25-36). Althoughthe proliferation targets for bantam have not been identified, apro-apoptotic gene, hid, was shown to have multiple bantam complementarysites in its 3′UTR. Since hid gene expression was repressed by thebantam miRNA, this implicates a role for bantam in controlling apoptosisby blocking hid function. Another Drosophila miRNA, mir-14, wasidentified in a genetic screen for factors that modified Reaper-inducedapoptosis in the fly eye (Xu, P., et al., Curr Biol (2003) 13(9):790-5).mir-14 was shown to be a strong suppressor of apoptosis. In addition,mir-14 also appears to play a role in the Drosophila stress response aswell as in regulating fat metabolism. MiRNAs also regulate Notch pathwaygenes in Drosophila (Lai, et al. Genes Dev (2005) 19(9):1067-80). Amammalian miRNA, mir-181, was shown to direct the differentiation ofhuman B cells (Chen, C. Z., et al., Science (2004) 303(5654): 83-6),mir-373 regulates insulin secretion (Poy, M. N., et al., Nature (2004)432(7014):226-30), while other miRNAs regulate viral infections(Lecellier, C. H., et al., Science (2005) 308(5721):557-;60 andSullivan, C. S., et al., Nature (2005) 435(7042):682-6).

Studies to understand the mechanism of RNAi in C. elegans, Drosophilaand human cells have shown that the miRNA and RNAi pathways mayintersect (Grishok, A., et al. Cell (2001) 106(1):23-34 and Hutvagner,G., et al., Science (2001) 293(5531):834-8). MiRNAs copurify withcomponents of the RNAi effector complex, RISC, suggesting a link betweenmiRNAs and siRNAs involved in RNAi (Mourelatos, Z., et al., Genes Dev(2002) 16(6):720-8; Hutvagner, G. and P. D. Zamore, Science (2002)297(5589):2056-60; and Caudy, A. A., et al., Nature (2003) 425(6956):411-4). There is also an indication that some protein factors may play arole in both the miRNA ribonucleoprotein (miRNP) and RISC and othersmight be unique to the miRNP (Grishok, A., et al., Cell (2001) 106(1):23-34 and Carmell, M. A., et al., Genes Dev (2002) 16(21): 2733-42). Forexample, proteins of the argonaute/PAZ/PIWI family are components ofboth RISC and miRNPs. There is also mounting evidence that genesencoding these proteins are linked to cancer. hAgo3, hAgo1, and hAgo4reside in region 1p34-35, often lost in Wilms' tumors, and Hiwi, islocated on chromosome 12q24.33, which has been linked to the developmentof testicular germ cell tumors (Carmell, M. A., et al., Genes Dev (2002)16(21):2733-42). In addition, DICER, the enzyme which processes miRNAsand siRNAs, is poorly expressed in lung cancers (Karube, Y., et al.,Cancer Sci (2005) 96(2):111-5).

It is therefore an object of the present invention to provide naturallyoccurring miRNAs for inhibition of expression of one or more oncogenes.

It is further an object of the present invention to provide naturallyoccurring nucleic acids for treatment or prophylaxis of one or moresymptoms of cancer.

BRIEF SUMMARY OF THE INVENTION

Genes that control cell differentiation and development are frequentlymutated in human cancers. These include, but are not limited to,oncogenes such as RAS, c-myc and bcl-2. Naturally occurring microRNAs,in particular let-7, have been found that down regulate these oncogenesin humans. Some of the let-7 genes are located in chromosomal regionsthat are deleted in certain cancers (FIG. 4). Therefore, up-regulatingthese specific microRNAs or providing analogous pharmaceutical compoundsexogenously, should be effective cancer therapies for tumors resultingfrom activation or over-expression of these oncogenes. MiRNAs nucleicacids including pri-miRNA, pre-miRNA, mature miRNA or fragments ofvariants thereof that retain the biological activity of the mature miRNAand DNA encoding a pri-miRNA, pre-miRNA, mature miRNA, fragments orvariants thereof, or regulatory elements of the miRNA, referred tojointly as “miRNAs” unless otherwise stated, are described. In oneembodiment, the size range of the miRNA can be from 21 nucleotides to170 nucleotides, although miRNAs of up to 2000 nucleotides can beutilized. In a preferred embodiment the size range of the miRNA is from70 to 170 nucleotides in length. In another preferred embodiment, maturemiRNAs of from 21 to 25 nucleotides in length can be used.

These miRNAs are useful as diagnostics and as therapeutics. Thecompositions are administered to a patient in need of treatment orprophylaxis of at least one symptom or manifestation (since disease canoccur/progress in the absence of symptoms) of cancer. Aberrantexpression of oncogenes is a hallmark of cancer, for example, lungcancer. In one embodiment, the compositions are administered in aneffective amount to inhibit expression of one or more oncogenes. In amore preferred embodiment, the compositions are administered in aneffective amount to inhibit expression of RAS, MYC, and/or BCL-2.Effective, safe dosages can be experimentally determined in modelorganisms and in human trials by methods well known to one of ordinaryskill in the art. The compositions can be administered alone or incombination with adjuvant cancer therapy such as surgery, chemotherapy,radiotherapy, thermotherapy, immunotherapy, hormone therapy and lasertherapy, to provide a beneficial effect, e.g. reduce tumor size, reducecell proliferation of the tumor, inhibit angiogenesis, inhibitmetastasis, or otherwise improve at least one symptom or manifestationof the disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the predicted secondary structure of the let-7 pre-miRNAfrom various organisms (SEO ID Nos. 1, 2 and 3). The shaded residuesindicate the mature miRNA transcript excised by Dicer.

FIG. 2 is a schematic of potential RNA/RNA duplexes between a C. elegansmiRNA, let-7, (SEQ ID No. 6) and the target mRNA, lin-41 (SEQ ID Nos. 4and 5). The position of a loss-of-function mutation for the let-7 miRNA,let-7(n2853), is shown by an arrow below the duplexes.

FIG. 3 depicts let-7 homologues from various species (SEQ ID Nos. 7, 8,9, 10, 11, 12., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25),including humans and mouse. There are 2 human homologues of lin-4 (SEQID No. 22), mir-125a (SEQ ID No. 25) and mir-125b (SEQ ID No. 23).mir-237 (SEQ ID No. 24) is a C. elegans lin-4 homologue.

FIGS. 4A-D depict potential LCSs in C. elegans let-60/RAS and inmammalian RAS genes. FIG. 4A C. elegans let-60/RAS mRNA 3′UTR, blackarrows indicate sites with similarity between C. elegans and C. briggsaeand white arrows indicate non-similar sites. Shown below are predictedduplexes formed by LCSs (SEQ ID Nos. 26, 27, 28, 29, 32, 33, 34 and 35)(top) and miR-84 (SEQ ID Nos. 30 and 31) (bottom). let-7 and miR-84 areso similar that most let-7 sites are also potential miR-84 sites. FIGS.4B, 4C, and 4D depict that H.s. NRAS, KRAS and HRAS mRNA 3′UTRs have 9(SEQ ID Nos. 36, 37, 38, 40, 41, 42, 43, 44 and 45), 8 (SEQ ID Nos. 46,47, 48, 49, 50, 51, 52 and 53) and 3 (SEQ ID Nos. 54, 55 and 56)potential LCSs, respectively. Black arrows indicate sites conservedamong mammalian species (in most cases human, rat, mouse, hamster andguinea pig). Shown below are hypothesized duplexes formed by (top) andlet-7a miRNA (SEQ ID No. 39) (bottom).

FIG. 5A is a graph of the quantitative analysis of the expressionpattern from five independent wild-type transgenic lines grown at 20° C.At least 25% repression was observed in all lines. A non-regulatedlin-41 3′UTR missing its LCSs (pFS1031), tested in duplicate is shown asa control. FIG. 5B is a graph depicting the down-regulation of thereporter gene expression is lost in let-7(n2853) mutant worms grown atthe permissive temperature, 15° C. The parental (N2) line was tested intriplicate; four isogenic let-7(n2853) mutant lines were tested. Errorbars represent standard deviations.

FIGS. 6A-C are graphs of the quantification of expression data. gfp54 isa fusion of gfp to the unc-54 3′UTR, driven by the lin-31 promoter.Error bars represent standard deviations.

FIGS. 7A and B are graphs showing that the presence of let-7 influencesthe expression of RAS in human cells. HEPG2 cells were transfected with10 and 30 nM of a let-7 or negative control precursor miRNA.Immunofluorescence using an antibody specific to NRAS, VRAS, and KRASrevealed that the let-7 transfected cells have much lower levels of theRAS proteins. FIG. 7A is a graph of the quantification of the RASantibody fluorescence from replicates of the transfections.Alternatively, HeLa cells were transfected with 100 nM let-7 inhibitoror negative control inhibitor. RAS immunofluorescence revealed thatcells transfected with the let-7 inhibitor has increased levels of theRAS proteins relative to the negative control transfected cells. FIG. 7Bis a graph of the quantification of the RAS antibody fluorescence fromreplicates of the transfections.

FIGS. 8A-C depict that the 3′UTRs of NRAS and KRAS enable let-7regulation. FIG. 8A is a schematic showing the NRAS short (NRAS S) NRASlong (NRAS L) and KRAS 3′UTRs. Arrows indicate LCSs. The blackened areasindicate the sequence cloned behind the reporter. FIG. 8B is a graph ofthe relative repression of firefly luciferase expression standardized toa transfection control, renilla luciferase. pGL3-Cont is the emptyvector. FIG. 8C is a graph of the induction of firefly luciferaseexpression when reporter plasmids with 3′UTR domains corresponding toKRAS and NRAS are co-transfected with an inhibitor of let-7, relative toa control inhibitor.

FIG. 9A is a graph of the expression of let-7 in 21 breast, colon, andlung tumors relative to associated normal adjacent tissue (NAT).Fluorescently labeled miRNA was hybridized to microarrays that includedprobes specific to let-7a and let-7c. Fluorescence intensities for thetumor and NAT were normalized by total fluorescence signal for allelements and the relative average signal from the let-7 probes in thetumor and normal adjacent samples are expressed as log ratios.

FIG. 9B is a graph of the correlation between RAS protein and let-7cexpression in tumor and normal adjacent tissue samples from three lungsquamous cell carcinomas. GAPDH and RAS proteins were measured fromcrude extracts of tumor and normal adjacent tissues using westernanalysis. The two proteins were assessed simultaneously by mixing theantibodies used for detection. The small RNA northern blot was assayedsequentially with radio-labeled probes specific to let-7c and U6 snRNA.NRAS mRNA in the tumor and normal adjacent tissues samples was measuredby real-time PCR. The real-time data were normalized based on thereal-time PCR detection of 18S rRNA in the various samples. The relativeexpression of NRAS in the normal adjacent tissues was taken to be 100%and the Ct value of NRAS in the tumor samples was used to assign therelative expression of NRAS in the tumor samples.

FIG. 10A is a sequence comparison of the let-7 family of miRNAs (SEQ IDNos. 19, 21, 58 and 59) in C. elegans. The Majority sequence isrepresented by SEQ ID No. 57. FIG. 10B is a dendrogram of let-7 familymembers.

FIG. 11 depicts potential LCSs (SEQ ID Nos. 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 and 80) in let-60/RASin C. elegans mRNA.

FIG. 12 is a sequence alignment of C. elegans (SEQ ID No. 81) (top) andC. briggsae (SEQ ID No. 82) (bottom) let-60/RAS mRNAs (consensussequence shown in middle) (SEQ ID No. 83). LCSs are shown as blackboxes.

FIGS. 13A-C show that Let-60 is the ortholog of human HRAS, KRAS, andNRAS proteins. FIG. 13A is a sequence alignment of C. elegans LET-60(SEQ ID No. 87) with other RAS and RAS related proteins from humans (SEQID Nos. 84, 85 and 86) and C. elegans (SEQ ID Nos. 88, 89, 90 and 91).FIG. 13B is a dendrogram showing the relationship of LET-60 to other RASrelated proteins. FIG. 13C is a graph showing quantification of thepartial suppression of let-60(gf) alleles. O84-X are linesoverexpressing mir-84, and TOPO-X are lines with the empty vectorcontrol. The average of each of the three experimental and control linesis indicated.

FIG. 14 is a sequence alignment of partial sequences from rodent (SEQ IDNos. 94, 95 and 96) and human (SEQ ID Nos. 92 and 93) NRAS 3′UTRs. TheLCSs shown in this alignment are boxed in black.

FIG. 15 (SEQ ID Nos. 39, 97, 98, 99, 100, 101, 102 and 103) depictspotential let-7::LCS duplexes formed with Xenopus laevis and Danio rerioNRAS 3′UTRs.

FIGS. 16A and B show that the presence of let-7 does not influence theexpression of control proteins in human cells. FIG. 16A is a graph ofthe quantification of the antibody fluorescence from replicates of HepG2cells transfected with let-7 or negative control siRNAs using antibodiesspecific to GAPDH or p21. FIG. 16B is a graph of the quantification offluorescent signal from a single field of 50-100 cells for both theNRAS- and Negative Control siRNA transfections.

FIG. 17 is a graph of the expression of let-7c and let-7g in lung tumorsrelative to associated normal adjacent tissue (NAT).

FIG. 18A is a graph of the inhibition of let-7 that results in a 100%increase of A549 cell numbers, compared to a control transfection and acontrol anti-miRNA (mir-19a). FIG. 18B is a graph of Extra let-7 thatcauses a decrease in A549 cell numbers compared to a control miRNA (NC).

FIG. 19 depicts potential let-7 complementary sites (boxed) in the 3′UTRof human MYC (SEQ ID No. 104) and other vertebrates' (SEQ ID Nos. 105,106 and 107) and potential duplexes between let-7 (SEQ ID No. 39 and110) and human MYC (SEQ ID Nos. 108 and 109).

FIG. 20A is a graph of the reduced expression of MYC and BCL-2 proteinin cells treated with exogenous let-7 miRNA compared to a control miRNAwith a scrambled let-7 sequence.

FIG. 20B is a graph of the increased expression of MYC and BCL-2 in HeLacells transfected with an anti-let-7 molecule.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein the term “nucleic acid” refers to multiple nucleotides(i.e. molecules comprising a sugar (e.g. ribose or deoxyribose) linkedto a phosphate group and to an exchangeable organic base, which iseither a substituted pyrimidine (e.g. cytosine (C), thymidine (T) oruracil (U)) or a substituted purine (e.g. adenine (A) or guanine (G)).The term shall also include polynucleosides (i.e. a polynucleotide minusthe phosphate) and any other organic base containing polymer. Purinesand pyrimidines include but are not limited to adenine, cytosine,guanine, thymidine, inosine, 5-methylcytosine, 2-aminopurine,2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, and othernaturally and non-naturally occurring nucleobases, substituted andunsubstituted aromatic moieties. Other such modifications are well knownto those of skill in the art. Thus, the term nucleic acid alsoencompasses nucleic acids with substitutions or modifications, such asin the bases and/or sugars.

“MicroRNA flanking sequence” as used herein refers to nucleotidesequences including microRNA processing elements. MicroRNA processingelements are the minimal nucleic acid sequences which contribute to theproduction of mature microRNA from precursor microRNA. Precursor miRNAtermed pri-miRNAs are processed in the nucleus into about 70 nucleotidepre-miRNAs, which fold into imperfect stem-loop structures.

The microRNA flanking sequences may be native microRNA flankingsequences or artificial microRNA flanking sequences. A native microRNAflanking sequence is a nucleotide sequence that is ordinarily associatedin naturally existing systems with microRNA sequences, i.e., thesesequences are found within the genomic sequences surrounding the minimalmicroRNA hairpin in vivo. Artificial microRNA flanking sequences arenucleotides sequences that are not found to be flanking to microRNAsequences in naturally existing systems. The artificial microRNAflanking sequences may be flanking sequences found naturally in thecontext of other microRNA sequences. Alternatively they may be composedof minimal microRNA processing elements which are found within naturallyoccurring flanking sequences and inserted into other random nucleic acidsequences that do not naturally occur as flanking sequences or onlypartially occur as natural flanking sequences.

The microRNA flanking sequences within the precursor microRNA moleculemay flank one or both sides of the stem-loop structure encompassing themicroRNA sequence. Preferred structures have flanking sequences on bothends of the stem-loop structure. The flanking sequences may be directlyadjacent to one or both ends of the stem-loop structure or may beconnected to the stem-loop structure through a linker, additionalnucleotides or other molecules.

As used herein a “stem-loop structure” refers to a nucleic acid having asecondary structure that includes a region of nucleotides which areknown or predicted to form a double strand (stem portion) that is linkedon one side by a region of predominantly single-stranded nucleotides(loop portion). The terms “hairpin” and “fold-back” structures are alsoused herein to refer to stem-loop structures. Such structures and termsare well known in the art. The actual primary sequence of nucleotideswithin the stem-loop structure is not critical as long as the secondarystructure is present. As is known in the art, the secondary structuredoes not require exact base-pairing. Thus, the stem may include one ormore base mismatches. Alternatively, the base-pairing may not includeany mismatches.

As used herein, the term “let-7” refers to the nucleic acid encoding thelet-7 miRNA and homologues and variants thereof including conservativesubstitutions, additions, and deletions therein not adversely affectingthe structure or function. Preferably, let-7 refers to the nucleic acidencoding let-7 from C. elegans (NCBI Accession No. AY390762), mostpreferably, let-7 refers to the nucleic acid encoding a let-7 familymember from humans, including but not limited to, NCBI Accession Nos.AJ421724, AJ421725, AJ421726, AJ421727, AJ421728, AJ421729, AJ421730,AJ421731, AJ421732, and biologically active sequence variants of let-7,including alleles, and in vitro generated derivatives of let-7 thatdemonstrate let-7 activity.

Sequence variants of let-7 fall into one or more of three classes:substitutional, insertional or deletional variants. Insertions include5′ and/or 3′ terminal fusions as well as intrasequence insertions ofsingle or multiple residues. Insertions can also be introduced withinthe mature sequence of let-7. These, however, ordinarily will be smallerinsertions than those at the 5′ or 3′ terminus, on the order of 1 to 4residues.

Insertional sequence variants of let-7 are those in which one or moreresidues are introduced into a predetermined site in the target let-7.Most commonly insertional variants are fusions of nucleic acids at the5′ or 3′ terminus of let-7.

Deletion variants are characterized by the removal of one or moreresidues from the let-7 RNA sequence. These variants ordinarily areprepared by site specific mutagenesis of nucleotides in the DNA encodinglet-7, thereby producing DNA encoding the variant, and thereafterexpressing the DNA in recombinant cell culture. However, variant let-7fragments may be conveniently prepared by in vitro synthesis. Thevariants typically exhibit the same qualitative biological activity asthe naturally-occurring analogue, although variants also are selected inorder to modify the characteristics of let-7.

Substitutional variants are those in which at least one residue sequencehas been removed and a different residue inserted in its place. Whilethe site for introducing a sequence variation is predetermined, themutation per se need not be predetermined. For example, in order tooptimize the performance of a mutation at a given site, randommutagenesis may be conducted at the target region and the expressedlet-7 variants screened for the optimal combination of desired activity.Techniques for making substitution mutations at predetermined sites inDNA having a known sequence are well known.

Nucleotide substitutions are typically of single residues; insertionsusually will be on the order of about from 1 to 10 residues; anddeletions will range about from 1 to 30 residues. Deletions orinsertions preferably are made in adjacent pairs; i.e. a deletion of 2residues or insertion of 2 residues. Substitutions, deletion, insertionsor any combination thereof may be combined to arrive at a finalconstruct. Changes may be made to increase the activity of the miRNA, toincrease its biological stability or half-life, and the like. All suchmodifications to the nucleotide sequences encoding such miRNA areencompassed.

A DNA isolate is understood to mean chemically synthesized DNA, cDNA orgenomic DNA with or without the 3′ and/or 5′ flanking regions. DNAencoding let-7 can be obtained from other sources by a) obtaining a cDNAlibrary from cells containing mRNA, b) conducting hybridization analysiswith labeled DNA encoding let-7 or fragments thereof (usually, greaterthan 100 bp) in order to detect clones in the cDNA library containinghomologous sequences, and c) analyzing the clones by restriction enzymeanalysis and nucleic acid sequencing to identify full-length clones.

As used herein nucleic acids and/or nucleic acid sequences arehomologous when they are derived, naturally or artificially, from acommon ancestral nucleic acid or nucleic acid sequence. Homology isgenerally inferred from sequence similarity between two or more nucleicacids or proteins (or sequences thereof). The precise percentage ofsimilarity between sequences that is useful in establishing homologyvaries with the nucleic acid and protein at issue, but as little as 25%sequence similarity is routinely used to establish homology. Higherlevels of sequence similarity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%,95% or 99% or more can also be used to establish homology. Methods fordetermining sequence similarity percentages (e.g., BLASTN using defaultparameters) are generally available. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (www.ncbi.nlm.nih.gov).

I. Compositions

Genes that control cell differentiation and development are frequentlymutated in human cancers. These include, but are not limited to,oncogenes such as RAS, c-myc and bcl-2. Naturally occurring microRNAs,in particular let-7, have been found that down regulate these oncogenesin humans. Some of the let-7 genes are located in chromosomal regionsthat are deleted in certain cancers. Therefore, up-regulating thesespecific microRNAs or providing analogous pharmaceutical compoundsexogenously, should be effective cancer therapies for tumors resultingfrom activation or over-expression of these oncogenes.

In preferred embodiments, the miRNA formulations are administered toindividuals with a cancer that expresses one or more targets of let-7 orlin-4. More preferably, the formulations are administered to individualswith a cancer that over expresses RAS, MYC and/or BCL-2 or other targethaving one or more binding sites for the let-7. Multiple differentpathways, along with the RAS/MAPK pathway, are implicated in cancer. The3′UTRs of many known cancer genes have been examined and potential let-7complementary sites have been identified in the 3′UTR of many of them(FIG. 19 and Table 1). These sites possess features of established let-7complementary sites in known C. elegans let-7 targets (Reinhart, et al.,Nature (2000) 403:901-906; Johnson, et al., Cell (2005) 120(5):635-47;Grosshans, et al., Dev Cell (2005) 8(3):321-30; Lin, et al., Dev Cell(2003) 4(5):639-50; Slack, et al., Molec. Cell (2000) 5:659-669; Vella,et al., Genes Dev (2004) 18(2):132-7). let-7 complementary sites (LCS)in the 3′UTRs of human c-MYC and BCL-2 have been identified (see FIG. 19for c-MYC). Many of the potential target genes shown in Table 1 are alsoup-regulated in lung cancer as well as other cancers, leading to theconclusion that let-7 is responsible for repressing their expression innormal tissues. Some genes, like GRB2, have a similar number of LCSs asknown let-7 target genes like KRAS (Table 1). VEGF was also identifiedas having let-7 complementary sites. These results indicate that let-7can repress expression of multiple oncogenes. In addition to inhibitingcell proliferation, let-7 may also inhibit angiogenesis. Therefore,administration of let-7 may inhibit multiple pathways that promotesurvival of cancer or tumor cells (i.e., angiongenesis, decreasedapoptosis and increased cell proliferation).

TABLE 1 Genes implicated in cancer that contain let-7 binding sites.Homo sapiens Gene Number of LCS sites EGF 1 EGFR 1 ERBB3 3 GRB2 10 NRAS9 KRAS2 8 HRAS 3 RAF1 1 ARAF 3 MAP2K2 2 MAPK1 1 MAPK3 4 MET 3 KIT 3TP73L(AIX) 5 MYC 2 MYCL1 6 MYCN 4 BCL2 5 BCL2L1 5 BCL2L2 6 CCND1/cyclinD3 CDK4 1 MDM2/HDM2 4 FES 2 FURIN 2 INSL3 2 CSF1R/FMS 1 MYBL2 1 MYB 1PIK3CD 6 PIK3C2B 4 PIK3CG 1 PIK3R5 1 TERT 3 AKT1 3 AKT3 2 VEGF 3 HLIN-414 VDR 7 PXR 3 FOXA1 2 FOXA2 A ASH1L 2 ARID1B 5 GR 2 GLI2 1 14-3-3zeta 6MO25 1 SMG1 2 FRAP1 3 PER2 4

miRNAs Have Known Roles in Human Cancer

Recent studies from different laboratories show roles for miRNAs inhuman cancer (McManus, Seminars in Cancer Biology (2003) 13:253-258).The human miRNAs, mir-15 and mir-16, are preferentially deleted ordown-regulated in patients with a common form of adult leukemia, B cellchronic lymphocytic leukemia (Calin, et al., Proc Natl Acad Sci USA(2002) 99(24): 15524-9). This study suggests that miRNAs may function astumor suppressor genes. The bic locus, which encodes the mir-155 miRNAworks cooperatively with c-myc and induces B-cell lymphomas, presumablyacting as a proto-oncogene (Haasch, D., et al., Cell Immunol (2002)217(1-2):78-86). miR142 acts as a tumor suppressor in chroniclymphocytic leukemia (Calin, G. A., et al., Proc Natl Acad Sci USA(2002) 99(24):15524-9; Lagos-Quintana, M., et al., Curr Biol (2002)12(9):735-9). His-1 acts as an oncogene in B cell lymphoma (Haasch, D.,et al., (2002); Lagos-Quintana, M., et al., (2002); Lagos-Quintana, M.,et al., Science (2001) 294(5543):853-8; Li, et al. Am J Pathol (1997)150:1297-305). Translocation of myc to the mir-142 locus causes B celllymphoma (Lagos-Quintana, M., et al., (2002); Gauwerky, C. E., et al.,Proc Natl Acad Sci USA, 1989. 86(22):8867-71). Mir-143 and mir-145 arepoorly expressed in colorectal cancer (Michael, M. Z., et al., MolCancer Res (2003) 1(12):882-91). Over-expression of the mir-17, 18, 19,20 locus is able to cause lymphomas in a mouse model and is up-regulatedby MYC (He, L., et al., Nature (2005) 435(7043):828-33; O'Donnell, K.A., et al., Nature (2005) 435(7043):839-43).

Misregulation of genes that control cell fate determination oftencontributes to cancer. Genes that control cell differentiation anddevelopment are frequently mutated in human cancer. The model organismCaenorhabditis elegans has been used to identify genes required for celldifferentiation in a stem-cell like lineage in the epidermis. C. elegansgrowth and development is divided into three major stages called embryo,larva and adult. Larval growth is subdivided into four larval stages(L1, L2, L3 and L4). Each larval stage ends in a molt and ultimately theanimal matures into an adult. The genes that regulate timing ofstage-appropriate cell division and differentiation are known asheterochronic genes (Slack, F. and G. Ruvkun, Annu Rev Genet (1997)31:611-34; Baneijee, D. and F. Slack, Bioessays (2002) 24(2): 119-29).In C. elegans, heterochronic genes control the timing of cell fatedetermination and differentiation. In heterochronic mutants, cellsfrequently fail to terminally differentiate, and instead divide again, ahallmark of cancer.

The founding members of the miRNA family, lineage defective-4 (lin-4)and lethal-7 (let-7), were identified through genetic analysis tocontrol the timing of stage-appropriate cell division anddifferentiation in C. elegans (Lee, et al. Cell (1993) 75(5):843-854;Reinhart, B., et al., Nature (2000) 403: 901-906; Slack, F. and G.Ruvkun, Annu Rev Genet (1997) 31: 611-34; Banerjee, D. and F. Slack,Bioessays (2002) 24(2): 119-29). let-7 and lin-4 control the timing ofproliferation versus differentiation decisions. Some of these genes,like lin-4 and let-7, encode microRNAs (miRNAs) that are conserved inhumans. Mutations in the lin-4 and let-7 miRNAs result in inappropriatereiterations of the first larval stage (L1) and the fourth larval stage(L4) fates, respectively, and these defects lead to disruptions in cellcycle exit.(Lee, et al. Cell (1993) 75(5):843-854; Reinhart, B., et al.,Nature (2000) 403:901-906). For example, in wild-type animals,specialized skin cells, known as seam cells, divide with a stem cellpattern and terminally differentiate at the beginning of the adultstage. The seam cells fail to terminally differentiate in lin-4 andlet-7 mutant animals, and instead reiterate the larval fate and divideagain. Lack of cell cycle control and failure to terminallydifferentiate are hallmarks of cancer.

The expression patterns for lin-4 and let-7 correlate with their role indirecting developmental timing. lin-4 RNA accumulates during the L1stage and is responsible for the L1/L2 transition in nematodes byinhibiting the expression of lin-14 and lin-28, repressors of post-L1fates (Lee, et al. Cell (1993) 75(5):843-854; Ambros, V. and H. R.Horvitz, Science (1984) 226:409-416; Wightman, et al. Cell (1993)75(5):855-862; Moss, et al. Cell (1997) 88(5): 37-46; and Feinbaum, R.and V. Ambros, Dev Biol (1999) 210(1):87-95). let-7 RNA accumulatesduring the L4 stage and is responsible for the L4/Adult transition bydown-regulating the expression of lin-41, hbl-1 and RAS (Johnson, etal., Cell (2005) 120(5):635-47; Grosshans, et al., Dev Cell (2005)8(3)321-30; Lin, et al., Dev Cell (2003) 4(5):639-50; Slack, F. J.,Molec. Cell (2000) 5:659-669).

These 21-22 nucleotide miRNAs exert their effect by binding to imperfectcomplementary sites within the 3′-untranslated regions (3′UTRs) of theirtarget protein-coding mRNAs and repress the expression of these genes atthe level of translation (Lee, et al. Cell (1993) 75(5):843-854;Reinhart, B., et al., Nature (2000) 403:901-906; Moss, et al. Cell(1997) 88(5):637-46; Lin, S. Y., et al., Dev Cell (2003) 4(5):639-50;Slack, F. J., et al., Molec. Cell (2000) 5:659-669; Abrahante, J. E., etal., Dev Cell (2003) 4(5):625-37; and Olsen, P. H. and V. Ambros, DevBiol (1999) 216(2):671-80). Deletion of let-7 miRNA complementary sites(LCS) (SEQ ID No. 4 and SEQ ID No. 5) from the lin-41 3′UTR (FIG. 2)showed abrogation of the normal down-regulation of lin-41 during the L4and adult stages and recent work has shown that these complementarysites alone are sufficient for regulation on lin-41 (Reinhart, B., etal., Nature (2000) 403:901-906; Slack, F. J., et al., Molec. Cell (2000)5:659-669; Vella, M.C., et al., Genes Dev (2004) 18(2):132-7).

The let-7 target gene, lin-41 is similar to known oncogenes. C. eleganslin-41 loss-of-function (lf) mutations cause cells to terminallydifferentiate precociously, opposite to of the effect seen withlet-7(lƒ), while over-expression of lin-41 causes let-7(lƒ)-like seamcell proliferation (Reinhart, B., et al., Nature (2000) 403: 901-906;Slack, F. J., et al., Molec. Cell (2000) 5:659-669). Like let-7, lin-41is an important cell proliferation and differentiation gene. let-7 andlin-41 work together to cause cells to proliferate or differentiate atthe right time.

lin-41 encodes a member of the RBCC (RING finger, B box, Coiled Coil(Freemont, Ann. New York Acad. Sci. (1993) 684:174-192) family ofproteins. Members of this family have diverse proposed functions, suchas transcription and RNA binding, and include the PML (Kakizuka, A., etal., Cell (1991) 66:663-674), TIF1 (Le Dourarin, B., et al., EMBO J.(1995) 14(9):2020-2033) and Rfp, proto-oncogenes. The most common formof promyelocytic leukemia involves a translocation that fuses PML to theRARa gene. The N-terminal part of TIF1, is fused to B-raf in theoncogenic protein T18 (Le Dourarin, B., et al., EMBO J. (1995)14(9):2020-2033). Emu-ret mice, carrying an RFP/RBT fusion gene underthe transcriptional control of the immunoglobulin heavy chain enhancer,develop B lineage leukemias and lymphomas (Wasserman, et al. Blood(1998) 92(1):273-82). In transformed NIH 3T3 cells, the amino-terminalhalf of Rfp with a RING finger motif is fused to a truncated Retreceptor tyrosine kinase, Rfp/Ret (Hasegawa, N., et al., Biochem BiophysRes Commun (1996) 225(2):627-31). Members of this family are associatedwith cancer progression. It is expected that mammalian lin-41 is also aproto-oncogene.

In addition to lin-41, let-7 regulates other target genes in a 3′ UTRdependent manner, including hunchback-like1 (hbl-1) (Lin Shin-Yi, J., etal. Dev. Cell, 2003(4): 1-20) and let-60, the C. elegans RAS oncogenehomologue (see Example 1). As shown in Table 2, let-60/RAS containsmultiple let-7 complimentary sites in its 3′UTR and let-60(lƒ)suppresses let-7 mutants. let-60/RAS is best understood for its role inC. elegans vulval development and let-60/RAS 3′UTR is sufficient torestrict let-60/RAS expression only to the vulval precursor cell (VPC)that absolutely requires let-60/ras activity (the primary induced cell,1° or P6.p cell). In a normal animal, a let-7 family member, mir-84, isexpressed in all the VPCs except the primary induced cell, and represseslet-60/ras expression in these cells.

In animals carrying let-60 activating mutations, more than one VPC isinduced to differentiate into the 1° cell fate, leading to excessvulvae. Over-expression of mir-84 suppresses activating mutations inlet-60/RAS. Many activating mutations in the human NRAS, KRAS and HRASgenes alter the same amino acid affected by the C. elegans let-60activating mutation. Since RAS is mutated in multiple human cancers(Malumbres, et al. Nat Rev Cancer (2003) 3(6):459-65), the hypothesisthat human RAS is a target of human let-7 was tested and determined tobe (see example 1 below).

lin-4 and let-7 miRNAs are evolutionarily conserved in higher animals,including humans (FIG. 3) (SEQ ID Nos. 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24 and 25) and temporally expressed (seeexamples below) which implies a universal role for these miRNAs duringanimal development (Lagos-Quintana, M., et al., Mouse. Curr Biol (2002)12(9):735-9 and Pasquinelli, A.E., et al., Nature (2000)408(6808):86-9). let-7 orthologues have been identified in mammals,including humans, and let-7 is expressed in human lung tissues as judgedby northern blot (Pasquinelli, A.E., et al.). There are 3 exact copiesof the mature let-7 sequence in the sequenced human genome (referred toas let-7a1, let-7a2, let-7a3 under control of separate promoters) and avariety of close homologues that differ from let-7 at certain nucleotidepositions (e.g. let-7c (SEQ ID No. 16), see FIG. 3). The nematode, flyand human let-7 genes are processed from a precursor form (pre-let-7)that is predicted to form a stem loop structure which is also conserved(FIG. 1). Similarly, there are 2 human and mouse homologues of lin-4(SEQ ID No. 22), named mir-125a (SEQ ID no. 25) and mir-125b (SEQ ID No.23) (FIG. 3).

Recent work has demonstrated that microRNA expression profiles canaccurately diagnose particular cancers better than standard messengerRNA expression profiles (Lu, et al., Nature 435:834-838 (2005)).

miRNAs Useful to Regulate Human Oncogenes

Naturally occurring microRNAs that regulate human oncogenes, pri-miRNA,pre-miRNA, mature miRNA or fragments of variants thereof that retain thebiological activity of the mature miRNA and DNA encoding a pri-miRNA,pre-miRNA, mature miRNA, fragments or variants thereof, or regulatoryelements of the miRNA, have been identified. The size of the miRNA istypically from 21 nucleotides to 170 nucleotides, although nucleotidesof up to 2000 nucleotides can be utilized. In a preferred embodiment thesize range of the pre-miRNA is between 70 to 170 nucleotides in lengthand the mature miRNA is between 21 and 25 nucleotides in length.

Nucleic Acids

General Techniques

General texts which describe molecular biological techniques includeSambrook, Molecular Cloning: a Laboratory Manual (2^(nd) ed.), Vols.1-3, Cold Spring Harbor Laboratory, (1989); Current Protocols inMolecular Biology, Ausubel, ed. John Wiley & Sons, Inc., New York(1997); Laboratory Techniques in Biochemistry and Molecular Biology:Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic AcidPreparation, P. Tijssen, ed. Elsevier, N.Y. (1993); Berger and Kimmel,Guide to Molecular Cloning Techniques Methods in Enzymology volume 152Academic Press, Inc., San Diego, Calif. These texts describemutagenesis, the use of vectors, promoters and many other relevanttopics related to, e.g., the generation and expression of genes thatencode let-7 or any other miRNA activity. Techniques for isolation,purification and manipulation of nucleic acids, genes, such asgenerating libraries, subcloning into expression vectors, labelingprobes, and DNA hybridization are also described in the texts above andare well known to one of ordinary skill in the art.

The nucleic acids, whether miRNA, DNA, cDNA, or genomic DNA, or avariant thereof, may be isolated from a variety of sources or may besynthesized in vitro. Nucleic acids as described herein can beadministered to or expressed in humans, transgenic animals, transformedcells, in a transformed cell lysate, or in a partially purified or asubstantially pure form.

Nucleic acids are detected and quantified in accordance with any of anumber of general means well known to those of skill in the art. Theseinclude, for example, analytical biochemical methods such asspectrophotometry, radiography, electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), and hyperdiffusion chromatography, variousimmunological methods, such as fluid or gel precipitin reactions,immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs),immuno-fluorescent assays, and the like, Southern analysis, Northernanalysis, Dot-blot analysis, gel electrophoresis, RT-PCR, quantitativePCR, other nucleic acid or target or signal amplification methods,radiolabeling, scintillation counting, and affinity chromatography.

Various types of mutagenesis can be used, e.g., to modify a nucleic acidencoding a gene with let-7 or other miRNA activity. They include but arenot limited to site-directed, random point mutagenesis, homologousrecombination (DNA shuffling), mutagenesis using uracil containingtemplates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, and mutagenesis using gappedduplex DNA or the like. Additional suitable methods include pointmismatch repair, mutagenesis using repair-deficient host strains,restriction-selection and restriction-purification, deletionmutagenesis, mutagenesis by total gene synthesis, double-strand breakrepair, and the like. Mutagenesis, e.g., involving chimeric constructs,are also included in the present invention. In one embodiment,mutagenesis can be guided by known information of the naturallyoccurring molecule or altered or mutated naturally occurring molecule,e.g., sequence, sequence comparisons, physical properties, crystalstructure or the like. Changes may be made to increase the activity ofthe miRNA, to increase its biological stability or half-life, and thelike.

Comparative hybridization can be used to identify nucleic acids encodinggenes with let-7 or other miRNA activity, including conservativevariations of nucleic acids.

Nucleic acids “hybridize” when they associate, typically in solution.Nucleic acids hybridize due to a variety of well characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology-Hybridization with Nucleic AcidProbes part 1 chapter 2, “Overview of principles of hybridization andthe strategy of nucleic acid probe assays,” (Elsevier, N.Y.), as well asin Ausubel, supra. Hames and Higgins (1995) Gene Probes 1 IRL Press atOxford University Press, Oxford, England, (Hames and Higgins 1) andHames and Higgins (1995) Gene Probes 2 IRL Press at Oxford UniversityPress, Oxford, England (Hames and Higgins 2) provide details on thesynthesis, labeling, detection and quantification of DNA and RNA,including oligonucleotides.

Nucleic acids which do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, e.g., when a copyof a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

Suitable nucleic acids for use in the methods described herein include,but are not limited to, pri-miRNA, pre-miRNA, mature miRNA or fragmentsof variants thereof that retain the biological activity of the miRNA andDNA encoding a pri-miRNA, pre-miRNA, mature miRNA, fragments or variantsthereof, or DNA encoding regulatory elements of the miRNA.

Viral Vectors

In one embodiment the nucleic acid encoding a miRNA molecule is on avector. These vectors include a sequence encoding a mature microRNA andin vivo expression elements. In a preferred embodiment, these vectorsinclude a sequence encoding a pre-miRNA and in vivo expression elementssuch that the pre-miRNA is expressed and processed in vivo into a maturemiRNA. In another embodiment, these vectors include a sequence encodingthe pri-miRNA gene and in vivo expression elements. In this embodiment,the primary transcript is first processed to produce the stem-loopprecursor miRNA molecule. The stem-loop precursor is then processed toproduce the mature microRNA.

Vectors include, but are not limited to, plasmids, cosmids, phagemids,viruses, other vehicles derived from viral or bacterial sources thathave been manipulated by the insertion or incorporation of the nucleicacid sequences for producing the microRNA, and free nucleic acidfragments which can be attached to these nucleic acid sequences. Viraland retroviral vectors are a preferred type of vector and include, butare not limited to, nucleic acid sequences from the following viruses:retroviruses, such as: Moloney murine leukemia virus; Murine stem cellvirus, Harvey murine sarcoma virus; murine mammary tumor virus; Roussarcoma virus; adenovirus; adeno-associated virus; SV40-type viruses;polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpesviruses; vaccinia viruses; polio viruses; and RNA viruses such as anyretrovirus. One of skill in the art can readily employ other vectorsknown in the art.

Viral vectors are generally based on non-cytopathic eukaryotic virusesin which non-essential genes have been replaced with the nucleic acidsequence of interest. Non-cytopathic viruses include retroviruses, thelife cycle of which involves reverse transcription of genomic viral RNAinto DNA with subsequent proviral integration into host cellular DNA.Retroviruses have been approved for human gene therapy trials.Genetically altered retroviral expression vectors have general utilityfor the high-efficiency transduction of nucleic acids in vivo. Standardprotocols for producing replication-deficient retroviruses (includingthe steps of incorporation of exogenous genetic material into a plasmid,transfection of a packaging cell lined with plasmid, production ofrecombinant retroviruses by the packaging cell line, collection of viralparticles from tissue culture media, and infection of the target cellswith viral particles) are provided in Kriegler, M., “Gene Transfer andExpression, A Laboratory Manual,” W.H. Freeman Co., New York (1990) andMurry, E. J. Ed. “Methods in Molecular Biology,” vol. 7, Humana Press,Inc., Cliffton, N.J. (1991).

Promoters

The “in vivo expression elements” are any regulatory nucleotidesequence, such as a promoter sequence or promoter-enhancer combination,which facilitates the efficient expression of the nucleic acid toproduce the microRNA. The in vivo expression element may, for example,be a mammalian or viral promoter, such as a constitutive or induciblepromoter or a tissue specific promoter. Examples of which are well knownto one of ordinary skill in the art. Constitutive mammalian promotersinclude, but are not limited to, polymerase promoters as well as thepromoters for the following genes: hypoxanthine phosphoribosyltransferase (HPTR), adenosine deaminase, pyruvate kinase, andbeta.-actin. Exemplary viral promoters which function constitutively ineukaryotic cells include, but are not limited to, promoters from thesimian virus, papilloma virus, adenovirus, human immunodeficiency virus(HIV), Rous sarcoma virus, cytomegalovirus, the long terminal repeats(LTR) of moloney leukemia virus and other retroviruses, and thethymidine kinase promoter of herpes simplex virus. Other constitutivepromoters are known to those of ordinary skill in the art. Induciblepromoters are expressed in the presence of an inducing agent andinclude, but are not limited to, metal-inducible promoters andsteroid-regulated promoters. For example, the metallothionein promoteris induced to promote transcription in the presence of certain metalions. Other inducible promoters are known to those of ordinary skill inthe art.

Examples of tissue-specific promoters include, but are not limited to,the promoter for creatine kinase, which has been used to directexpression in muscle and cardiac tissue and immunoglobulin heavy orlight chain promoters for expression in B cells. Other tissue specificpromoters include the human smooth muscle alpha-actin promoter.

Exemplary tissue-specific expression elements for the liver include butare not limited to HMG-COA reductase promoter, sterol regulatory element1, phosphoenol pyruvate carboxy kinase (PEPCK) promoter, humanC-reactive protein (CRP) promoter, human glucokinase promoter,cholesterol 7-alpha hydroylase (CYP-7) promoter, beta-galactosidasealpha-2,6 sialyltransferase promoter, insulin-like growth factor bindingprotein (IGFBP-1) promoter, aldolase B promoter, human transferrinpromoter, and collagen type I promoter.

Exemplary tissue-specific expression elements for the prostate includebut are not limited to the prostatic acid phosphatase (PAP) promoter,prostatic secretory protein of 94 (PSP 94) promoter, prostate specificantigen complex promoter, and human glandular kallikrein gene promoter(hgt-1).

Exemplary tissue-specific expression elements for gastric tissue includebut are not limited to the human H+/K+-ATPase alpha subunit promoter.

Exemplary tissue-specific expression elements for the pancreas includebut are not limited to pancreatitis associated protein promoter (PAP),elastase 1 transcriptional enhancer, pancreas specific amylase andelastase enhancer promoter, and pancreatic cholesterol esterase genepromoter.

Exemplary tissue-specific expression elements for the endometriuminclude, but are not limited to, the uteroglobin promoter.

Exemplary tissue-specific expression elements for adrenal cells include,but are not limited to, cholesterol side-chain cleavage (SCC) promoter.

Exemplary tissue-specific expression elements for the general nervoussystem include, but are not limited to, gamma-gamma enolase(neuron-specific enolase, NSE) promoter.

Exemplary tissue-specific expression elements for the brain include, butare not limited to, the neurofilament heavy chain (NF-H) promoter.

Exemplary tissue-specific expression elements for lymphocytes include,but are not limited to, the human CGL-1/granzyme B promoter, theterminal deoxy transferase (TdT), lambda 5, VpreB, and lck (lymphocytespecific tyrosine protein kinase p56lck) promoter, the humans CD2promoter and its 3′ transcriptional enhancer, and the human NK and Tcell specific activation (NKG5) promoter.

Exemplary tissue-specific expression elements for the colon include, butare not limited to, pp60c-src tyrosine kinase promoter, organ-specificneoantigens (OSNs) promoter, and colon specific antigen-P promoter.

Exemplary tissue-specific expression elements for breast cells include,but are not limited to, the human alpha-lactalbumin promoter.

Exemplary tissue-specific expression elements for the lung include, butare not limited to, the cystic fibrosis transmembrane conductanceregulator (CFTR) gene promoter.

Other elements aiding specificity of expression in a tissue of interestcan include secretion leader sequences, enhancers, nuclear localizationsignals, endosmolytic peptides, etc. Preferably, these elements arederived from the tissue of interest to aid specificity.

In general, the in vivo expression element shall include, as necessary,5′ non-transcribing and 5′ non-translating sequences involved with theinitiation of transcription. They optionally include enhancer sequencesor upstream activator sequences.

Methods and Materials for Production of miRNA

The miRNA can be isolated from cells or tissues, recombinantly produced,or synthesized in vitro by a variety of techniques well known to one ofordinary skill in the art.

In one embodiment, miRNA is isolated from cells or tissues. Techniquesfor isolating miRNA from cells or tissues are well known to one ofordinary skill in the art. For example, miRNA can be isolated from totalRNA using the mirVana miRNA isolation kit from Ambion, Inc. Anothertechniques utilizes the flashPAGE™ Fractionator System (Ambion, Inc.)for PAGE purification of small nucleic acids.

The miRNA can be obtained by preparing a recombinant version thereof(i.e., by using the techniques of genetic engineering to produce arecombinant nucleic acid which can then be isolated or purified bytechniques well known to one of ordinary skill in the art). Thisembodiment involves growing a culture of host cells in a suitableculture medium, and purifying the miRNA from the cells or the culture inwhich the cells are grown. For example, the methods include a processfor producing a miRNA in which a host cell containing a suitableexpression vector that includes a nucleic acid encoding an miRNA iscultured under conditions that allow expression of the encoded miRNA. Ina preferred embodiment the nucleic acid encodes let-7. The miRNA can berecovered from the culture, from the culture medium or from a lysateprepared from the host cells, and further purified. The host cell can bea higher eukaryotic host cell such as a mammalian cell, a lowereukaryotic host cell such as a yeast cell, or the host cell can be aprokaryotic cell such as a bacterial cell. Introduction of a vectorcontaining the nucleic acid encoding the miRNA into the host cell can beeffected by calcium phosphate transfection, DEAE, dextran mediatedtransfection, or electroporation (Davis, L. et al., Basic Methods inMolecular Biology (1986)).

Any host/vector system can be used to express one or more of the miRNAs.These include, but are not limited to, eukaryotic hosts such as HeLacells and yeast, as well as prokaryotic host such as E. coli and B.subtilis. miRNA can be expressed in mammalian cells, yeast, bacteria, orother cells where the miRNA gene is under the control of an appropriatepromoter. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al., inMolecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y. (1989). In the preferred embodiment, the miRNA is expressedin mammalian cells. Examples of mammalian expression systems includeC127, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney293 cells, human epidermal A43 1 cells, human Colo205 cells, 3T3 cells,CV-1 cells, other transformed primate cell lines, normal diploid cells,cell strains derived from in vitro culture of primary tissue, primaryexplants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkatcells. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter, polyadenylation site, transcriptionaltermination sequences, and 5′ flanking nontranscribed sequences. DNAsequences derived from the SV40 viral genome, for example, SV40 origin,early promoter, enhancer, splice, and polyadenylation sites may be usedto provide the required nontranscribed genetic elements. Potentiallysuitable yeast strains include Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeaststrain capable of expressing miRNA. Potentially suitable bacterialstrains include Escherichia coli, Bacillus subtilis, Salmonellatyphimurium, or any bacterial strain capable of expressing miRNA.

In a preferred embodiment, genomic DNA encoding let-7 is isolated, thegenomic DNA is expressed in a mammalian expression system, RNA ispurified and modified as necessary for administration to a patient. In apreferred embodiment the let-7 is in the form of a pre-miRNA, which canbe modified as desired (i.e. for increased stability or cellularuptake).

Knowledge of DNA sequences of miRNA allows for modification of cells topermit or increase expression of an endogenous miRNA. Cells can bemodified (e.g., by homologous recombination) to provide increased miRNAexpression by replacing, in whole or in part, the naturally occurringpromoter with all or part of a heterologous promoter so that the cellsexpress the miRNA at higher levels. The heterologous promoter isinserted in such a manner that it is operatively linked to the desiredmiRNA encoding sequences. See, for example, PCT InternationalPublication No. WO 94/12650 by Transkaryotic Therapies, Inc., PCTInternational Publication No. WO 92/20808 by Cell Genesys, Inc., and PCTInternational Publication No. WO 91/09955 by Applied Research Systems.Cells also may be engineered to express an endogenous gene comprisingthe miRNA under the control of inducible regulatory elements, in whichcase the regulatory sequences of the endogenous gene may be replaced byhomologous recombination. Gene activation techniques are described inU.S. Pat. No. 5,272,071 to Chappel; U.S. Pat. No. 5,578,461 to Sherwinet al.; PCT/US92/09627 (WO93/09222) by Selden et al.; and PCT/US90/06436(WO91/06667) by Skoultchi et al.

The miRNA may be prepared by culturing transformed host cells underculture conditions suitable to express the miRNA. The resultingexpressed miRNA may then be purified from such culture (i.e., fromculture medium or cell extracts) using known purification processes,such as gel filtration and ion exchange chromatography. The purificationof the miRNA may also include an affinity column containing agents whichwill bind to the protein; one or more column steps over such affinityresins as concanavalin A-agarose, heparin-toyopearl™ or Cibacrom blue3GA Sepharose™; one or more steps involving hydrophobic interactionchromatography using such resins as phenyl ether, butyl ether, or propylether; immunoaffinity chromatography, or complementary cDNA affinitychromatography.

The miRNA may also be expressed as a product of transgenic animals,which are characterized by somatic or germ cells containing a nucleotidesequence encoding the miRNA. A vector containing DNA encoding miRNA andappropriate regulatory elements can be inserted in the germ line ofanimals using homologous recombination (Capecchi, Science 244:1288-1292(1989)), such that the express the miRNA. Transgenic animals, preferablynon-human mammals, are produced using methods as described in U.S. Pat.No 5,489,743 to Robinson, et al., and PCT Publication No. WO 94/28122 byOntario Cancer Institute. miRNA can be isolated from cells or tissueisolated from transgenic animals as discussed above.

In a preferred embodiment, the miRNA can be obtained synthetically, forexample, by chemically synthesizing a nucleic acid by any method ofsynthesis known to the skilled artisan. The synthesized miRNA can thenbe purified by any method known in the art. Methods for chemicalsynthesis of nucleic acids include, but are not limited to, in vitrochemical synthesis using phosphotriester, phosphate or phosphoramiditecheminstry and solid phase techniques, or via deosynucleosideH-phosphonate intermediates (see U.S. Pat. No. 5,705,629 to Bhongle).

In some circumstances, for example, where increased nuclease stabilityis desired, nucleic acids having nucleic acid analogs and/or modifiedinternucleoside linkages may be preferred. Nucleic acids containingmodified internucleoside linkages may also be synthesized using reagentsand methods that are well known in the art. For example, methods ofsynthesizing nucleic acids containing phosphonate phosphorothioate,phosphorodithioate, phosphoramidate methoxyethyl phosphoramidate,formacetal, thioformacetal, diisopropylsilyl, acetamidate, carbamate,dimethylene-sulfide (—CH₂—S—CH₂), diinethylene-sulfoxide (—CH₂—SO—CH₂),dimethylene-sulfone (—CH₂—SO₂—CH₂), 2′-O-alkyl, and 2′-deoxy-2′-fluorophosphorothioate internucleoside linkages are well known in the art (seeUhlmann et al., 1990, Chem. Rev. 90:543-584; Schneider et al., 1990,Tetrahedron Lett. 31:335 and references cited therein). U.S. Pat. No.5,614,617 and 5,223,618 to Cook, et al., U.S. Pat. No. 5,714,606 toAcevedo, et al., U.S. Pat. No. 5,378,825 to Cook, et al., U.S. Pat. No.5,672,697 and 5,466,786 to Buhr, et al., U.S. Pat. No. 5,777,092 toCook, et al., U.S. Pat. No. 5,602,240 to De Mesmaeker, et al., U.S. Pat.No. 5,610,289 to Cook, et al. and U.S. Pat. No. 5,858,988 to Wang, alsodescribe nucleic acid analogs for enhanced nuclease stability andcellular uptake.

Formulations

The compositions are administered to a patient in need of treatment orprophylaxis of at least one symptom or manifestation (since disease canoccur/progress in the absence of symptoms) of cancer. Aberrantexpression of oncogenes is a hallmark of cancer. In a preferredembodiment, the cancer is lung cancer. In one embodiment, thecompositions are administered in an effective amount to inhibit geneexpression of one or more oncogenes. In preferred embodiments, thecompositions are administered in an effective amount to inhibit geneexpression of RAS; MYC, and/or BCL-2.

Methods for treatment or prevention of at least one symptom ormanifestation of cancer are also described consisting of administrationof an effective amount of a composition containing a nucleic acidmolecule to alleviate at least one symptom or decrease at least onemanifestation. In a preferred embodiment, the cancer is lung cancer. Thecompositions described herein can be administered in effective dosagesalone or in combination with adjuvant cancer therapy such as surgery,chemotherapy, radiotherapy, thermotherapy, immunotherapy, hormonetherapy and laser therapy, to provide a beneficial effect, e.g. reducetumor size, reduce cell proliferation of the tumor, inhibitangiogenesis, inhibit metastasis, or otherwise improve at least onesymptom or manifestation of the disease.

The nucleic acids described above are preferably employed fortherapeutic uses in combination with a suitable pharmaceutical carrier.Such compositions comprise an effective amount of the compound, and apharmaceutically acceptable carrier or excipient. The formulation ismade to suit the mode of administration. Pharmaceutically acceptablecarriers are determined in part by the particular composition beingadministered, as well as by the particular method used to administer thecomposition. Accordingly, there is a wide variety of suitableformulations of pharmaceutical compositions containing the nucleic acidssome of which are described herein.

It is understood by one of ordinary skill in the art that nucleic acidsadministered in vivo are taken up and distributed to cells and tissues(Huang, et al., FEBS Lett. 558(1-3):69-73 (2004)). For example, Nyce etal. have shown that antisense oligodeoxynucleotides (ODNs) when inhaledbind to endogenous surfactant (a lipid produced by lung cells) and aretaken up by lung cells without a need for additional carrier lipids(Nyce and Metzger, Nature, 385:721-725 (1997). Small nucleic acids arereadily taken up into T24 bladder carcinoma tissue culture cells (Ma, etal., Antisense Nucleic Acid Drug Dev. 8:415-426 (1998). siRNAs have beenused for therapeutic silencing of an endogenous genes by systemicadministration (Soutschek, et al., Nature 432, 173-178 (2004)).

The nucleic acids described above may be in a formulation foradministration topically, locally or systemically in a suitablepharmaceutical carrier. Remington's Pharmaceutical Sciences, 15thEdition by E. W. Martin (Mark Publishing Company, 1975), disclosestypical carriers and methods of preparation. The nucleic acids may alsobe encapsulated in suitable biocompatible microcapsules, microparticlesor microspheres formed of biodegradable or non-biodegradable polymers orproteins or liposomes for targeting to cells. Such systems are wellknown to those skilled in the art and may be optimized for use with theappropriate nucleic acid.

Various methods for nucleic acid delivery are described, for example inSambrook et al., 1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York; and Ausubel et al., 1994, CurrentProtocols in Molecular Biology, John Wiley & Sons, New York. Suchnucleic acid delivery systems comprise the desired nucleic acid, by wayof example and not by limitation, in either “naked” form as a “naked”nucleic acid, or formulated in a vehicle suitable for delivery, such asin a complex with a cationic molecule or a liposome forming lipid, or asa component of a vector, or a component of a pharmaceutical composition.The nucleic acid delivery system can be provided to the cell eitherdirectly, such as by contacting it with the cell, or indirectly, such asthrough the action of any biological process. By way of example, and notby limitation, the nucleic acid delivery system can be provided to thecell by endocytosis, receptor targeting, coupling with native orsynthetic cell membrane fragments, physical means such aselectroporation, combining the nucleic acid delivery system with apolymeric carrier such as a controlled release film or nanoparticle ormicroparticle, using a vector, injecting the nucleic acid deliverysystem into a tissue or fluid surrounding the cell, simple diffusion ofthe nucleic acid delivery system across the cell membrane, or by anyactive or passive transport mechanism across the cell membrane.Additionally, the nucleic acid delivery system can be provided to thecell using techniques such as antibody-related targeting andantibody-mediated immobilization of a viral vector.

Formulations for topical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like can be used as desired.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions, solutions or emulsions thatcan include suspending agents, solubilizers, thickening agents,dispersing agents, stabilizers, and preservatives. Formulations forinjection may be presented in unit dosage form, e.g., in ampules or inmulti-dose containers, with an added preservative. The compositions maytake such forms as.

Preparations include sterile aqueous or nonaqueous solutions,suspensions and emulsions, which can be isotonic with the blood of thesubject in certain embodiments. Examples of nonaqueous solvents arepolypropylene glycol, polyethylene glycol, vegetable oil such as oliveoil, sesame oil, coconut oil, arachis oil, peanut oil, mineral oil,injectable organic esters such as ethyl oleate, or fixed oils includingsynthetic mono or di-glycerides. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles include sodium chloridesolution, 1,3-butandiol, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers (such as thosebased on Ringer's dextrose), and the like. Preservatives and otheradditives may also be present such as, for example, antimicrobials,antioxidants, chelating agents and inert gases and the like. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or di-glycerides. In addition, fattyacids such as oleic acid may be used in the preparation of injectables.Carrier formulation can be found in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa. Those of skill in the art can readilydetermine the various parameters for preparing and formulating thecompositions without resort to undue experimentation.

The nucleic acids alone or in combination with other suitablecomponents, can also be made into aerosol formulations (i.e., they canbe “nebulized”) to be administered via inhalation. Aerosol formulationscan be placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. Foradministration by inhalation, the nucleic acids are convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant.

In some embodiments, the nucleic acids described above may includepharmaceutically acceptable carriers with formulation ingredients suchas salts, carriers, buffering agents, emulsifiers, diluents, excipients,chelating agents, fillers, drying agents, antioxidants, antimicrobials,preservatives, binding agents, bulking agents, silicas, solubilizers, orstabilizers. In one embodiment, the nucleic acids are conjugated tolipophilic groups like cholesterol and lauric and lithocholic acidderivatives with C32 functionality to improve cellular uptake. Forexample, cholesterol has been demonstrated to enhance uptake and serumstability of siRNA in vitro (Lorenz, et al., Bioorg. Med. Chem. Lett.14(19):4975-4977 (2004)) and in vivo (Soutschek, et al., Nature432(7014):173-178 (2004)). In addition, it has been shown that bindingof steroid conjugated oligonucleotides to different lipoproteins in thebloodstream, such as LDL, protect integrity and facilitatebiodistribution (Rump, et al., Biochem. Pharmacol. 59(11):1407-1416(2000)). Other groups that can be attached or conjugated to the nucleicacids described above to increase cellular uptake, include, but are notlimited to, acridinederivatives; cross-linkers such as psoralenderivatives, azidophenacyl, proflavin, and azidoproflavin; artificialendonucleases; metal complexes such as EDTA-Fe(II) and porphyrin-Fe(II);alkylating moieties,; nucleases such as alkaline phosphatase; terminaltransferases; abzymes; cholesteryl moieties; lipophilic carriers;peptide conjugates; long chain alcohols; phosphate esters; radioactivemarkers; non-radioactive markers; carbohydrates; and polylysine or otherpolyamines. U.S. Pat. No. 6,919,208 to Levy, et al., also describedmethods for enhanced delivery of nucleic acids molecules.

These pharmaceutical formulations may be manufactured in a manner thatis itself known, e.g., by means of conventional mixing, dissolving,granulating, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes.

The formulations described herein of the nucleic acids embrace fusionsof the nucleic acids or modifications of the nucleic acids, wherein thenucleic acid is fused to another moiety or moieties, e.g., targetingmoiety or another therapeutic agent. Such analogs may exhibit improvedproperties such as activity and/or stability. Examples of moieties whichmay be linked or unlinked to the nucleic acid include, for example,targeting moieties which provide for the delivery of nucleic acid tospecific cells, e.g., antibodies to pancreatic cells, immune cells, lungcells or any other preferred cell type, as well as receptor and ligandsexpressed on the preferred cell type. Preferably, the moieties targetcancer or tumor cells. For example, since cancer cells have increasedconsumption of glucose, the nucleic acids can be linked to glucosemolecules. Monoclonal humanized antibodies that target cancer or tumorcells are preferred moieties and can be linked or unlinked to thenucleic acids. In the case of cancer therapeutics, the target antigen istypically a protein that is unique and/or essential to the tumor cells(e.g., the receptor protein HER-2).

II. Methods of Treatment

Method of Administration

In general, methods of administering nucleic acids are well known in theart. In particular, the routes of administration already in use fornucleic acid therapeutics, along with formulations in current use,provide preferred routes of administration and formulation for thenucleic acids described above.

Nucleic acid compositions can be administered by a number of routesincluding, but not limited to: oral, intravenous, intraperitoneal,intramuscular, transdermal, subcutaneous, topical, sublingual, or rectalmeans. Nucleic acids can also be administered via liposomes. Suchadministration routes and appropriate formulations are generally knownto those of skill in the art.

Administration of the formulations described herein may be accomplishedby any acceptable method which allows the miRNA or nucleic acid encodingthe miRNA to reach its target. The particular mode selected will dependof course, upon factors such as the particular formulation, the severityof the state of the subject being treated, and the dosage required fortherapeutic efficacy. As generally used herein, an “effective amount” ofa nucleic acids is that amount which is able to treat one or moresymptoms of cancer or related disease, reverse the progression of one ormore symptoms of cancer or related disease, halt the progression of oneor more symptoms of cancer or related disease, or prevent the occurrenceof one or more symptoms of cancer or related disease in a subject towhom the formulation is administered, as compared to a matched subjectnot receiving the compound or therapeutic agent. The actual effectiveamounts of drug can vary according to the specific drug or combinationthereof being utilized, the particular composition formulated, the modeof administration, and the age, weight, condition of the patient, andseverity of the symptoms or condition being treated.

Any acceptable method known to one of ordinary skill in the art may beused to administer a formulation to the subject. The administration maybe localized (i.e., to a particular region, physiological system,tissue, organ, or cell type) or systemic, depending on the conditionbeing treated.

Injections can be e.g., intravenous, intradermal, subcutaneous,intramuscular, or intraperitoneal. The composition can be injectedintradermally for treatment or prevention of cancer, for example. Insome embodiments, the injections can be given at multiple locations.Implantation includes inserting implantable drug delivery systems, e.g.,microspheres, hydrogels, polymeric reservoirs, cholesterol matrixes,polymeric systems, e.g., matrix erosion and/or diffusion systems andnon-polymeric systems, e.g., compressed, fused, or partially-fusedpellets. Inhalation includes administering the composition with anaerosol in an inhaler, either alone or attached to a carrier that can beabsorbed. For systemic administration, it may be preferred that thecomposition is encapsulated in liposomes.

Preferably, the agent and/or nucleic acid delivery system are providedin a manner which enables tissue-specific uptake of the agent and/ornucleic acid delivery system. Techniques include using tissue or organlocalizing devices, such as wound dressings or transdermal deliverysystems, using invasive devices such as vascular or urinary catheters,and using interventional devices such as stents having drug deliverycapability and configured as expansive devices or stent grafts.

The formulations may be delivered using a bioerodible implant by way ofdiffusion or by degradation of the polymeric matrix. In certainembodiments, the administration of the formulation may be designed so asto result in sequential exposures to the miRNA over a certain timeperiod, for example, hours, days, weeks, months or years. This may beaccomplished, for example, by repeated administrations of a formulationor by a sustained or controlled release delivery system in which themiRNA is delivered over a prolonged period without repeatedadministrations. Administration of the formulations using such adelivery system may be, for example, by oral dosage forms, bolusinjections, transdermal patches or subcutaneous implants. Maintaining asubstantially constant concentration of the composition may be preferredin some cases.

Other delivery systems suitable include, but are not limited to,time-release, delayed release, sustained release, or controlled releasedelivery systems. Such systems may avoid repeated administrations inmany cases, increasing convenience to the subject and the physician.Many types of release delivery systems are available and known to thoseof ordinary skill in the art. They include, for example, polymer-basedsystems such as polylactic and/or polyglycolic acids, polyanhydrides,polycaprolactones, copolyoxalates, polyesteramides, polyorthoesters,polyhydroxybutyric acid, and/or combinations of these. Microcapsules ofthe foregoing polymers containing nucleic acids are described in, forexample, U.S. Pat. No. 5,075,109. Other examples include nonpolymersystems that are lipid-based including sterols such as cholesterol,cholesterol esters, and fatty acids or neutral fats such as mono-, di-and triglycerides; hydrogel release systems; liposome-based systems;phospholipid based-systems; silastic systems; peptide based systems; waxcoatings; compressed tablets using conventional binders and excipients;or partially fused implants. Specific examples include, but are notlimited to, erosional systems in which the miRNA is contained in aformulation within a matrix (for example, as described in U.S. Pat. Nos.4,452,775, 4,675,189, 5,736,152, 4,667,013, 4,748,034 and 5,239,660), ordiffusional systems in which an active component controls the releaserate (for example, as described in U.S. Pat. Nos. 3,832,253, 3,854,480,5,133,974 and 5,407,686). The formulation may be as, for example,microspheres, hydrogels, polymeric reservoirs, cholesterol matrices, orpolymeric systems. In some embodiments, the system may allow sustainedor controlled release of the composition to occur, for example, throughcontrol of the diffusion or erosion/degradation rate of the formulationcontaining the miRNA. In addition, a pump-based hardware delivery systemmay be used to deliver one or more embodiments.

Examples of systems in which release occurs in bursts includes, e.g.,systems in which the composition is entrapped in liposomes which areencapsulated in a polymer matrix, the liposomes being sensitive tospecific stimuli, e.g., temperature, pH, light or a degrading enzyme andsystems in which the composition is encapsulated by an ionically-coatedmicrocapsule with a microcapsule core degrading enzyme. Examples ofsystems in which release of the inhibitor is gradual and continuousinclude, e.g., erosional systems in which the composition is containedin a form within a matrix and effusional systems in which thecomposition permeates at a controlled rate, e.g., through a polymer.Such sustained release systems can be e.g., in the form of pellets, orcapsules.

Use of a long-term release implant may be particularly suitable in someembodiments. “Long-term release,” as used herein, means that the implantcontaining the composition is constructed and arranged to delivertherapeutically effective levels of the composition for at least 30 or45 days, and preferably at least 60 or 90 days, or even longer in somecases. Long-term release implants are well known to those of ordinaryskill in the art, and include some of the release systems describedabove.

Dosages for a particular patient can be determined by one of ordinaryskill in the art using conventional considerations, (e.g. by means of anappropriate, conventional pharmacological protocol). A physician may,for example, prescribe a relatively low dose at first, subsequentlyincreasing the dose until an appropriate response is obtained. The doseadministered to a patient is sufficient to effect a beneficialtherapeutic response in the patient over time, or, e.g., to reducesymptoms, or other appropriate activity, depending on the application.The dose is determined by the efficacy of the particular formulation,and the activity, stability or serum half-life of the miRNA employed andthe condition of the patient, as well as the body weight or surface areaof the patient to be treated. The size of the dose is also determined bythe existence, nature, and extent of any adverse side-effects thataccompany the administration of a particular vector, formulation, or thelike in a particular patient.

Therapeutic compositions comprising one or more nucleic acids areoptionally tested in one or more appropriate in vitro and/or in vivoanimal models of disease, to confirm efficacy, tissue metabolism, and toestimate dosages, according to methods well known in the art. Inparticular, dosages can be initially determined by activity, stabilityor other suitable measures of treatment vs. non-treatment (e.g.,comparison of treated vs. untreated cells or animal models), in arelevant assay. Formulations are administered at a rate determined bythe LD50 of the relevant formulation, and/or observation of anyside-effects of the nucleic acids at various concentrations, e.g., asapplied to the mass and overall health of the patient. Administrationcan be accomplished via single or divided doses.

In vitro models can be used to determine the effective doses of thenucleic acids as a potential cancer treatment. Suitable in vitro modelsinclude, but are not limited to, proliferation assays of cultured tumorcells, growth of cultured tumor cells in soft agar (see Freshney, (1987)Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, NewYork, N.Y. Ch 18 and Ch 21), tumor systems in nude mice as described inGiovanella et al., J. Natl. Can. Inst., 52: 921-30 (1974), mobility andinvasive potential of tumor cells in Boyden Chamber assays as describedin Pilkington et al., Anticancer Res., 17: 4107-9 (1997), andangiogenesis assays such as induction of vascularization of the chickchorioallantoic membrane or induction of vascular endothelial cellmigration as described in Ribatta et al., Intl. J. Dev. Biol., 40:1189-97 (1999) and Li et al., Clin. Exp. Metastasis, 17:423-9 (1999),respectively. Suitable tumor cells lines are available, e.g. fromAmerican Type Tissue Culture Collection catalogs.

In vivo models are the preferred models to determine the effective dosesof nucleic acids described above as potential cancer treatments.Suitable in vivo models include, but are not limited to, mice that carrya mutation in the KRAS oncogene (Lox-Stop-Lox K-Ras^(G12D) mutants,Kras2^(tm4TYj)) available from the National Cancer Institute (NCI)Frederick Mouse Repository. Other mouse models known in the art and thatare available include but are not limited to models for gastrointestinalcancer, hematopoietic cancer, lung cancer, mammary gland cancer, nervoussystem cancer, ovarian cancer, prostate cancer, skin cancer, cervicalcancer, oral cancer, and sarcoma cancer (seehttp://emice.nci.nih.gov/mouse_models/).

In determining the effective amount of the miRNA to be administered inthe treatment or prophylaxis of disease the physician evaluatescirculating plasma levels, formulation toxicities, and progression ofthe disease.

The dose administered to a 70 kilogram patient is typically in the rangeequivalent to dosages of currently-used therapeutic antisenseoligonucleotides such as Vitravene® (fomivirsen sodium injection) whichis approved by the FDA for treatment of cytomegaloviral RNA, adjustedfor the altered activity or serum half-life of the relevant composition.

The formulations described herein can supplement treatment conditions byany known conventional therapy, including, but not limited to, antibodyadministration, vaccine administration, administration of cytotoxicagents, natural amino acid polypeptides, nucleic acids, nucleotideanalogues, and biologic response modifiers. Two or more combinedcompounds may be used together or sequentially. For example, the nucleicacids can also be administered in therapeutically effective amounts as aportion of an anti-cancer cocktail. An anti-cancer cocktail is a mixtureof the oligonucleotide or modulator with one or more anti-cancer drugsin addition to a pharmaceutically acceptable carrier for delivery. Theuse of anti-cancer cocktails as a cancer treatment is routine.Anti-cancer drugs that are well known in the art and can be used as atreatment in combination with the nucleic acids described hereininclude, but are not limited to: Actinomycin D, Aminoglutethimide,Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine,Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine HCl(Cytosine arabinoside), Dacarbazine, Dactinomycin, Daunorubicin HCl,Doxorubicin HCl, Estramustine phosphate sodium, Etoposide (V16-213),Floxuridine, 5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea(hydroxycarbamide), Ifosfamide, Interferon Alpha-2a, InterferonAlpha-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine,Mechlorethamine HCl (nitrogen mustard), Melphalan, Mercaptopurine,Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCl, Octreotide,Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate,Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate,Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2, Mitoguazone,Pentostatin, Semustine, Teniposide, and Vindesine sulfate.

Diseases to be Treated

Cancer treatments promote tumor regression by inhibiting tumor cellproliferation, inhibiting angiogenesis (growth of new blood vessels thatis necessary to support tumor growth) and/or prohibiting metastasis byreducing tumor cell motility or invasiveness. Therapeutic formulationsdescribed herein may be effective in adult and pediatric oncologyincluding in solid phase tumors/malignancies, locally advanced tumors,human soft tissue sarcomas, metastatic cancer, including lymphaticmetastases, blood cell malignancies including multiple myeloma, acuteand chronic leukemias, and lymphomas, head and neck cancers includingmouth cancer, larynx cancer and thyroid cancer, lung cancers includingsmall cell carcinoma and non-small cell cancers, breast cancersincluding small cell carcinoma and ductal carcinoma, gastrointestinalcancers including esophageal cancer, stomach cancer, colon cancer,colorectal cancer and polyps associated with colorectal neoplasia,pancreatic cancers, liver cancer, urologic cancers including bladdercancer and prostate cancer, malignancies of the female genital tractincluding ovarian carcinoma, uterine (including endometrial) cancers,and solid tumor in the ovarian follicle, kidney cancers including renalcell carcinoma, brain cancers including intrinsic brain tumors,neuroblastoma, astrocytic brain tumors, gliomas, metastatic tumor cellinvasion in the central nervous system, bone cancers including osteomas,skin cancers including malignant melanoma, tumor progression of humanskin keratinocytes, squamous cell carcinoma, basal cell carcinoma,hemangiopericytoma and Karposi's sarcoma. Therapeutic formulations canbe administered in therapeutically effective dosages alone or incombination with adjuvant cancer therapy such as surgery, chemotherapy,radiotherapy, thermotherapy, immunotherapy, hormone therapy and lasertherapy, to provide a beneficial effect, e.g. reducing tumor size,slowing rate of tumor growth, reducing cell proliferation of the tumor,promoting cancer cell death, inhibiting angiongenesis, inhibitingmetastasis, or otherwise improving overall clinical condition, withoutnecessarily eradicating the cancer.

Cancers include, but are not limited to, biliary tract cancer; bladdercancer; breast cancer; brain cancer including glioblastomas andmedulloblastomas; cervical cancer; choriocarcinoma; colon cancerincluding colorectal carcinomas; endometrial cancer; esophageal cancer;gastric cancer; head and neck cancer; hematological neoplasms includingacute lymphocytic and myelogenous leukemia, multiple myeloma,AIDS-associated leukemias and adult T-cell leukemia lymphoma;intraepithelial neoplasms including Bowen's disease and Paget's disease;liver cancer; lung cancer including small cell lung cancer and non-smallcell lung cancer; lymphomas including Hodgkin's disease and lymphocyticlymphomas; neuroblastomas; oral cancer including squamous cellcarcinoma; osteosarcomas; ovarian cancer including those arising fromepithelial cells, stromal cells, germ cells and mesenchymal cells;pancreatic cancer; prostate cancer; rectal cancer; sarcomas includingleiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, synovialsarcoma and osteosarcoma; skin cancer including melanomas, Kaposi'ssarcoma, basocellular cancer, and squamous cell cancer; testicularcancer including germinal tumors such as seminoma, non-seminoma(teratomas, choriocarcinomas), stromal tumors, and germ cell tumors;thyroid cancer including thyroid adenocarcinoma and medullar carcinoma;transitional cancer and renal cancer including adenocarcinoma and Wilmstumor. In a preferred embodiment, the formulations are administered fortreatment or prevention of lung cancer.

In addition, therapeutic nucleic acids may be used for prophylactictreatment of cancer. There are hereditary conditions and/orenvironmental situations (e.g. exposure to carcinogens) known in the artthat predispose an individual to developing cancers. Under thesecircumstances, it may be beneficial to treat these individuals withtherapeutically effective doses of the nucleic acids to reduce the riskof developing cancers. In one embodiment, a nucleic acid in a suitableformulation may be administered to a subject who has a family history ofcancer, or to a subject who has a genetic predisposition for cancer. Inother embodiments, the nucleic acid in a suitable formulation isadministered to a subject who has reached a particular age, or to asubject more likely to get cancer. In yet other embodiments, the nucleicacid in a suitable formulation is administered to subjects who exhibitsymptoms of cancer (e.g., early or advanced). In still otherembodiments, the nucleic acid in a suitable formulation may beadministered to a subject as a preventive measure. In some embodiments,the nucleic acid in a suitable formulation may be administered to asubject based on demographics or epidemiological studies, or to asubject in a particular field or career.

EXAMPLES

The present invention will be further understood by reference to thefollowing non-limiting examples.

Example 1 RAS is Regulated by the let-7 microRNA Family

C. elegans let-7, mir-48, mir-84 and mir-241 encode four developmentallyregulated miRNAs that comprise the let-7 family (Lau et al., (2001)Science 294, 858-862; Lim et al., (2003) Genes Dev 17, 991-1008;Reinhart et al., (2000) Nature 403, 901-906). This family displays highsequence identity, with particular conservation at the 5′end of themature miRNAs (FIG. 10A, 10B). The C. elegans let-7 family miRNAs,mir-84, plays a role in vulval development, a model for dissectingRAS/MAP Kinase. signaling (Wang and Sternberg, (2001) Curr Top Dev Biol51, 189-220). C. elegans let-60/RAS is regulated by members of the let-7family. let-7 and mir-84 are complementary to multiple sites in the 3′UTR of let-601RAS. let-7 and mir-84 are expressed in a reciprocal mannerto let-60/RAS in the hypodermis and the vulva respectively.

let-7 and mir-84 genetically interact with let-60/RAS, consistent withnegative regulation of RAS expression by let-7 and mir-84. The resultsdemonstrate for the first time that miRNAs can regulate RAS, a criticalhuman oncogene. Moreover, all three human RAS genes have let-7complementary sites in their 3′UTRs that subject the oncogenes to let-7miRNA-mediated regulation in cell culture. Lung tumor tissues displaysignificantly reduced levels of let-7 and significantly increased levelsof RAS protein relative to normal lung tissue, suggesting let-7regulation of RAS as a mechanism for let-7 in lung oncogenesis.

Experimental Procedures

Plasmid Constructs. PSJ840 (mir-84::gfp) was made by amplifying 2.2 kbof genomic sequence (base pairs −2201 to −9) upstream of the maturemir-84 sequence from N2 genomic DNA and adding a SmaI site and an AgeIsite to the 5

and 3

ends, respectively, using the polymerase chain reaction (PCR) withprimers MIR84UP and MIR84DN (all primer sequences available uponrequest). This product was digested with SmaI and AgeI and then clonedinto the pPD95.70 vector digested with SmaI and AgeI. The mir-84upstream DNA contained various sequence elements that are also found inthe let-7 upstream DNA. PSJo84 (mir-84(+++)) was made by amplifying 3.0kb of genomic sequence (base pairs −2201 to +792) upstream anddownstream of the mature mir-84 sequence including the mir-84 sequenceitself from N2 genomic DNA and adding SmaI sites to both the 5

and 3

ends using PCR with primers MIR84UP and MIR84DN2. This product wasligated into the pCR4-TOPO vector using the TOPO TA Cloning Kit asdescribed by the manufacturer (Invitrogen). The empty TOPO controlvector was made by digesting PSJo84 with EcoRI, extracting the 4 kbvector band and self ligating it. The miR-84 deletion plasmid, o84Δ84(Δmir-84(+++)), was made by overlap extension PCR, starting with twoseparate PCR reactions using primers MIR84UP with DEL84DN and MIR84DN2with DEL84UP and PSJo84 plasmid as template. The two PCR products werepurified (QIAGEN) and then used together as template for the final PCRusing primers MIR84UP and MIR84DN2. This final product, identical toPSJo84 except for the deletion of the 75 nt pre-miR84 encoding sequence,was ligated into the pCR2.1-TOPO vector using the TOPO TA Cloning Kit(Invitrogen) and called o84Δ84. GFP60 was made by amplifying the 819 bpof genomic sequence encoding the let-60 3

UTR from N2 genomic DNA and adding EcoRI and SpeI sites to the 5

and 3

ends, respectively, using PCR with primers 3LET60UP and 3LET60DN. Thisproduct was digested with EcoRI and SpeI and then cloned into thepPD95.70 vector (Fire Lab) digested with EcoRI and SpeI, thus replacingthe unc-54 3

UTR found in pPD95.70 with the let-60 3

UTR. The green fluorescent protein gene (GFP) followed by the let-60 3

UTR was then amplified out of this plasmid and BglII and NotI sites wereadded to the 5

and 3

ends respectively using PCR with primers BGLGFP and 3UTRNOT. This PCRproduct was digested with BglII and NotI and ligated into PB255 digestedwith BglII and NotI, resulting in the plasmid GFP60 containing thelin-31 promoter and enhancer driving GFP with the let-60 3

UTR. GFP54 was made by amplifying gfp followed by the unc-54 3

UTR out of pPD95.70 using PCR with primers BGLGFP and 3UTRNOT. This PCRproduct was digested and ligated into PB255 digested with BglII andNotI, resulting in the plasmid GFP54 containing the lin-31 promoter andenhancer driving GFP with the unc-54 3

UTR. pGL3-NRAS S and pGL3-NRAS L were made by amplifying the entire 1140bp shorter form of the H.s. NRAS 3

UTR out of a IMAGE cDNA clone (accession # AI625442), or the entire 3616bp longer form of the H.s. NRAS 3

UTR (excluding the first 43 bp) from H.s. genomic DNA, and adding NheIsites to the ends by PCR using primers SMJ100 and SMJ101 or SMJ102 andSMJ103 respectively. These products were digested with NheI and ligatedinto pGL3-Control (Promega) digested with XbaI and treated with CIP,resulting in Rrluc-expressing plasmids containing either the short orlong form of the H.s. NRAS 3

UTR. To generate pFS1047, containing the col-10-lacZ-let-60 3

UTR reporter gene, the entire let-60 3

UTR was subcloned into the SacH and NcoI sites of plasmid B29 (Wightman,B., Ha, I., and Ruvkun, G. (1993) Cell 75, 855-862).

let-60; let-7 Double Mutants. let-60(n2021); let-7(mn112) double mutantanimals were generated by crossing let-60(n2021) heterozygotes withlet-7(mn112) unc-3/0; mnDp1 hemiyzgotes. From this cross, 180 individualhomozygous let-7(mn112) F2 animals were female, as determined by theirUnc-phenotype, due to the tight linkage of the let-7(mn112) to unc-3. Ofthese, 10 survived into adulthood and produced eggs. The resultingprogeny from these animals died as larvae with the rod-like phenotypethat is characteristic of let-60 mutant animals, thus showing that theseanimals all contained the let-60(n2021) mutation, and that adultsurvival was likely due to the let-60(n2021) mutation, confirming ourRNAi data. From 180 F2s, 25% (or about 45 animals) would have beenpredicted to be let-60(n2021) homozygous. Since one saw survival of only10 of these animals, this indicates a suppression of about 22%, similarto what was observed with RNAi. However, because the brood sizes werelow (usually about 10 hatched larvae and several unhatched eggs); andbecause of a combination of larval lethality as well as limited parentalsurvival, double mutant lines could not be established for furtheranalysis; all progeny died as larvae.

C. elegans and Transgenic reporter analysis. All animal experiments wereperformed at room temperature or 20° C. unless stated otherwise. Allexperimental plasmids were injected in animals at 50-100 ng/μl. Twodifferent markers, rol-6 (100 ng/μl) and myo-3:: gfp (50 ng/μl), wereseparately co-injected with PSJo84: myo-3:: gfp (50 ng/μl) wasco-injected with o84Δ84; and myo-2:: gfp (5 ng/μl) was co-injected withGFP60 and GFP54. These animals are mosaic for the transgenes. To compareexpression between individual lines, the percent expression of GFP ineach of the Pn.p cells was normalized relative to the expression of thehighest expressing Pn.p cell and represented as a fraction of thehighest expresser for each individual line of animals. For eachconstruct the average of the lines was calculated along with thestandard deviation for each construct represented as error bars(mir-84:: gfp n=239, gfp60 n=42 and gfp54 n=40). For the mir-84(+++)analysis, animals were examined using DIC optics to score seam cell andvulval anatomy. LacZ reporter analysis was as described (Vella et al.,(2004) Genes Dev 18, 132-137). The lin-41 3′UTR missing its LCSs(pFS1031) was used as a control (Reinhart et al., (2000)). RNAi methodswere standard feeding procedures using synchronized L1s (Timmons et al.,(2001) Gene 263, 103-112). All RNAi experiments were done in parallel toan empty vector (L4440) feeding control. See Supplemental Experimentalprocedures for details on the let-60; let-7 double mutant cross.

let-71RAS association in mammalian cells. HeLa S3 cells grown in D-MEM(GIBCO) supplemented with 10% fetal bovine serum (GIBCO) werecotransfected in 12-well plates using Lipofectamine 2000 (Invitrogen)according to the manufacturer's protocol using 1.0 μg/well ofPp-luc-expressing plasmid (pGL3-Control from Promega, pGL3-NRAS SpGL3-NRAS L and pGL3-KRAS) and 0.1 μg/well of Rr-luc-expressing plasmid(pRL-TK from Promega). 24 hrs post transfection, the cells wereharvested and assayed using the Dual Luciferase assay as described bythe manufacturer (Promega). HeLa cells grown as above were transfectedin 24-well plates with 30 pmoles of Anti-miR let-7 or negative control#1 inhibitors (Ambion) using Lipofectamine 2000. Three dayspost-transfection, RAS expression was monitored by immunofluorescenceusing an FITC conjugated primary antibody against RAS protein (USBiological). The resulting fluorescent signal was analyzed using theappropriate filter set and was quantified using MetaMorph software. Thefluorescence intensity of 150-300 cells was typically measured in one ora few viewing areas. The experiments with both the precursors and theinhibitors were performed three times. The photos represent singleviewing fields from one of the experiments and are representative of thetriplicate experiment. Identically grown HeLa cells were cotransfectedin 24-well plates using Lipofectamine 2000 (Invitrogen) according to themanufacturer's protocol using 200 ng /well of Pp-luc-expressing plasmid(pGL3-Control from Promega, pGL3-NRAS S pGL3-NRAS L and pGL3-KRAS). 48hrs post transfection, the cells were harvested and assayed using theLuciferase assay as described by the manufacturer (Promega).

HepG2 cells grown in D-MBM (GIBCO) supplemented with 10% fetal bovineserum (GIBCO) were transfected with 15 or 5 pmole of Pre-miR Let-7c ornegative control #1 Precursor miRNAs (Ambion) in 24-well plates usingsiPort Neo-FX (Ambion) according to the manufacturer's protocol. Threedays post-transfection, RAS expression was monitored byimmunofluorescence as described above.

MiRNA microarray analysis procedures used by Ambion, Inc. Total RNA fromtumor and normal adjacent tissue (NAT) samples from 3 breast cancer, 6colon cancer, and 12 lung cancer patients was isolated using the mirVanaRNA Isolation Kit (Ambion). Twenty μg of each total RNA sample wasfractionated by polyacrylamide gel electrophoresis (PAGE) using a 15%denaturing polyacrylamide gel and the miRNA fractions for each samplewere recovered. The miRNAs from all of the samples were subjected to apoly(A) polymerase reaction wherein amine modified uridines wereincorporated as part of 40 nt long tails (Ambion). The tailed tumorsamples were fluorescently labeled using an amine-reactive Cy3(Amersham) and the normal adjacent tissue samples were labeled with Cy5(Amersham). The fluorescently labeled miRNAs were purified byglass-fiber filter binding and elution (Ambion) and the tumor and normaladjacent tissue samples from the same patient were mixed. Each samplemixture was hybridized for 14 hours with slides upon which 167 miRNAprobes were arrayed. The microarrays were washed 3×2 minutes (min) in2×SSC and scanned using a GenePix 4000B (Axon). Fluorescence intensitiesfor the Cy3- and Cy5-labeled samples for each element were normalized bytotal Cy3 and Cy5 signal on the arrays. The normalized signal intensityfor each element was compared between the tumor and NAT samples fromeach pair of patient samples and expressed as a log ratio of the tumorto normal adjacent sample.

Northern Analysis. mir-84 northerns were performed as described (Johnsonet al., (2003) Dev Biol 259, 364-379). For human tissues, 1 ug of totalRNA from the tumor and normal adjacent tissues of patients 1 and 5 (FIG.9) were fractionated by PAGE using a 15% denaturing polyacrylamide gel.The RNA was transferred to a positively charged nylon membrane byelectroblotting at 200 mA in 0.5× TBE for 2 hours. The Northern blot wasdried and then incubated overnight in 10 ml of ULTRAhyb-Oligo (Ambion)with 10⁷ cpm of a radio-labeled transcript complementary to let-7c. Theblot was washed 3×10 min at room temperature in 2×SSC, 0.5% SDS and then1×15 min at 42° C. in 2×SSC, 0.5% SDS. Overnight phosphorimaging usingthe Storm system (Amersham), revealed let-7c. The process was repeatedusing a radio-labeled probe for 5S rRNA.

Lung tumor protein/northern/mRNA analysis. Total RNA and protein wereisolated from tumor and normal adjacent tissue samples from three lungcancer patients using the mirVana PARIS Kit (Ambion). let-7 miRNA and U6snRNA were measured using the Northern procedure described above. NRASand B-actin mRNA as well as 18S rRNA were quantified by real-time RT-PCRusing primers specific to each of the target RNAs. RAS and GAPDH proteinwere measured by Western analysis using the RAS antibody described aboveand an antibody for GAPDH (Ambion).

Accession numbers. The following sequences were searched for 3′UTR LCSs:Hs KRAS (Genbank M54968), Hs HRAS (NM176795), Hs NRAS (BC005219). NRASis known to exist as a 2 Kb and a 4.3 Kb form. BC005219 represents theshort form with a 1151 nt polyadenylated 3′UTR. Two human EST clones(Genbank BU177671 and BG388501) were sequenced to obtain additional NRAS3′UTR sequence. This revealed that the NRAS 3′UTR exists in a 3642 ntpolyadenylated 3′UTR version, utilizing an alternative polyadenylationand cleavage site, 2491 nt downstream of the first. This presumablycorresponds to the long NRAS form. The sequence was deposited withaccession numbers AY941100 and AY941101.

Results

Additional targets including let-60/RAS of the let-7 miRNA in C.elegans. The let-7 miRNA is temporally expressed in C. elegans (Johnsonet al., (2003) Nature 426, 845-849; Pasquinelli et al., (2000) Nature408, 86-89; Reinhart et al., (2000) Nature 403, 901-906) where itdown-regulates at least two target genes, lin-41 (Slack et al., (2000)Molec Cell 5, 659-669) and hb1-1 (Abrahante et al., (2003) Dev Cell 4,625-637; Lin et al., (2003) Dev Cell 4, 639-650), mutations which leadto precocious terminal differentiation of seam cells. To betterunderstand the role of let-7 in C. elegans seam cell differentiation andits potential role in humans, additional targets of let-7 wereidentified. A computational screen for C. elegans genes was performedwith let-7 family complementary sites (LCS) in their 3′UTR (Grosshans,H., et al., Dev Cell (2005) 8(3):321-30). One of the top scoring geneswas let-60, encoding the C. elegans orthologue of the human oncogeneRAS. 8 LCSs were identified in the 3′UTR of let-60 with featuresresembling validated LCSs (Lin et al., (2003) Dev Cell 4, 639-650;Reinhart et al., (2000) Nature 403, 901-906; Slack et al., (2000) MolecCell 5, 659-669; Vella et al., (2004) Genes Dev 18, 132-137) (FIG. 4A)(SEQ ID Nos. 26, 27, 28, 29, 32, 33, 34 and 35). Many of the identifiedsites were found in the 3′UTR of let-60 from the closely relatednematode C. briggsae (Stein et al., (2003) PLoS Biol 1, E45) (FIG. 4A ,FIG. 11 and FIG. 12), suggesting that they are likely to be biologicallysignificant. An additional three sites were found in the let-60/RAScoding sequence as well as 10 other non-conforming 3′UTR sites that mayalso bind to let-7 family miRNAs (FIG. 11) (SEQ ID Nos. 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 and 80).

let-7(n2853ts) loss of function (lf) mutants express reduced let-7 miRNAand die by bursting at the vulva at the non-permissive temperature(Reinhart et al., (2000); Slack et al., (2000) Molec Cell 5, 659-669).Loss of function mutations in two previously identified targets oflet-7, lin-41 and hbl-1, have the property of partially suppressing thelet-7 lethal phenotype (Abrahante et al., (2003) Dev Cell 4, 625-637;Lin et al., (2003) Dev Cell 4, 639-650; Reinhart et al., (2000) Nature403, 901-906; Slack et al., (2000) Molec Cell 5, 659-669). It was foundthat post-embryonic reduction of function of let-60 by feeding RNAinterference (RNAi), also partially suppressed let-7(n2853) in areproducible manner. While 5% of let-7 mutants grown on control RNAisurvived at the non-permissive temperature of 25° C. (n=302), 27% oflet-7(n2853); let-60(RNAi) animals survived (n=345). Thus, similar toother known let-7 targets, let-60 If partially suppresses thelet-7(n2853) lethal phenotype, suggesting that let-7 lethality may atleast partially be caused by over-expression of let-60. Howeverlet-60(RNAi) did not appear to suppress the let-7 seam cell terminaldifferentiation defect and did not cause precocious seam cell terminaldifferentiation. In addition, wild-type animals subjected tolet-60(RNAi) did not display typical lethal and vulvaless phenotypesassociated with let-60 alleles (Beitel et al., (1990) Nature 348,503-509; Han et al., (1990) Genetics 126, 899-913; Han and Sternberg,(1990) Cell 63, 921-931). let-60(RNAi) resulted in approximately 80%knock down of let-60 mRNA (Grosshans, H., et al., (2005) Dev Cell8(3):321-30), suggesting that the remaining let-60 is still sufficientfor seam cell differentiation and vulval development. To verify thespecificity of the let-60(RNAi), it was shown that while let-7(mn112)adults all die, let-60(n2021); let-7(mn112) adults can live (seeExperimental Procedures). Interestingly, let-7(n2853); let-60(RNAi)animals delivered a brood and could lay some eggs, suggesting that thevulval bursting phenotype of let-7 was not suppressed merely because ofthe lack of a vulva.

let-60 and let-7 are both expressed in hypodermal seam cells (Dent andHan, 1998 Post-embryonic expression pattern of C. elegans let-60 rasreporter constructs. Mech Dev 72, 179-182; Johnson et al., 2003 AmicroRNA controlling left/right neuronal asymmetry in Caenorhabditiselegans. Nature 426, 845-849). The let-60 3′UTR was fused behind theEscherichia coli lacZ gene driven by the hypodermally-expressing col-10promoter. It was found that reporter gene activity is down-regulatedaround the L4 stage (FIG. 5A), around the same time that let-7 isexpressed in the seam cells (Johnson et al., (2003) Nature 426,845-849). In contrast, the same reporter gene fused to an unregulatedcontrol 3′UTR was expressed at all stages (FIG. 5A) (Reinhart et al.,(2000) Nature 403, 901-906; Slack et al., (2000) Molec Cell 5, 659-669;Vella et al., (2004) Genes Dev 18, 132-137; Wightman et al., (1993) Cell75, 855-862). It was found that reporter down-regulation directed by thelet-60 3′UTR depended on a wild-type let-7 gene, since down-regulationfailed in let-7(n2853) mutants (FIG. 5B). Thus, multiple lines ofevidence strongly suggest that let-60 is negatively regulated by let-7.First, the let-60 3′UTR contains multiple elements complementary tolet-7; second, the let-60 3′UTR directs down-regulation of a reportergene in a let-7 dependent manner; third, this down-regulation isreciprocal to let-7 up-regulation in the hypodermis; and finally, let-60loss of function partially suppresses the let-7 lethal phenotype.

The let-7 family member mir-84 is dynamically expressed in the vulvalprecursor cells. let-60/RAS is best understood for its role in vulvaldevelopment (Wang and Sternberg, (2001) Curr Top Dev Biol 51, 189-220),however let-7 has not been reported to be expressed in the vulva. In C.elegans, let-7, mir-48, mir-84 and mir-241 comprise the let-7 family(Lau et al., (2001) Science 294, 858-862; Lim et al., (2003) Genes Dev17, 991-1008; Reinhart et al., (2000) Nature 403, 901-906) (FIGS. 10A,10B). Previous work demonstrated that a let-7:: gfp fusion faithfullyrecapitulates the temporal expression of let-7 and is temporallyexpressed in seam cell tissues affected in the let-7 mutant (Johnson etal., (2003) Dev Biol 259, 364-379). The expression pattern of mir-84,the closest let-7 relative, was examined by fusing 2.2 kilobases (Kb) ofgenomic sequence immediately upstream of the miR-84 encoding sequence tothe green fluorescent protein (gfp) gene. mir-84:: gfp was firstobserved in the somatic gonad in larval stage 1 (L1). In L3 animals,strong expression was observed in uterine cells including the anchorcell (AC), and weak dynamic expression was observed in the vulvalprecursor cells (VPCs) (FIGS. 6A-C). VPCs are multipotent ventralhypodermal cells that generate the vulva during L3 and later stages(Sulston and Horvitz, (1977) Dev Biol 56, 110-156). VPCs adopt one ofthree fates depending on EGF signaling from the AC (Wang and Sternberg,(2001) Curr Top Dev Biol 51, 189-220). The cell closest to the AC, P6.p,receives the most LIN-3/EGF signal (Katz et al., (1995) Cell 82,297-307) and adopts the primary (1⁰) fate through activation of aRAS/MAPK signal transduction pathway (Beitel et al., (1990) Nature 348,503-509; Han et al., (1990) Genetics 126, 899-913; Han and Sternberg,(1990) Cell 63, 921-931): P5.p and P7.p receive less LIN-3 as well asreceiving a secondary lateral signal (Sternberg, (1988) Nature 335,551-554) from P6.p, and adopt the secondary (2⁰) fate: P3.p, P4.p andP8.p adopt the uninduced tertiary (3⁰) fate. mir-84:: gfp expression wasobserved during the early to mid L3 stage in all the VPCs except forP6.p, in which expression was rarely observed (FIG. 6A). Subsequent VPCexpression in the mid to late L3 stage was restricted to the daughters(Pn.px) of P5.p and P7.p with weaker GFP first appearing in the P6.pdaughters just before their division into P6.pxx. Thereafter, equivalentexpression was observed in the granddaughters (Pn.pxx) of P5.p, P6.p andP7.p. mir-84:: gfp expression was observed in all the VPCs except forP6.p at the stage when their fate in vulval development is determined bysignaling from the AC (Ambros, (1999) Development 126, 1947-1956)suggesting that mir-84 could play a role in vulval cell fatedetermination. In the L4 stage; GFP expression was maintained in the ACand other uterine cells, appeared weakly in hypodermal seam cells, andwas up-regulated to higher levels in many P5.p-P7.p descendants. Asecond let-7 family member, mir-48 was also expressed in non-P6.p VPCs,suggesting the potential for redundancy between mir-48 and mir-84 in theVPCs.

mir-84 overexpression causes vulval and seam defects. miR-84 wasoverexpressed by generating transgenic animals harboring a multi-copyarray of a 3.0 Kb genomic DNA fragment that spans from 2.2 Kb upstreamto 0.8 Kb downstream of the miR-84 encoding sequence (calledmir-84(+++)). These animals expressed elevated levels of miR-84 anddisplayed abnormal vulval development phenotypes, including protrusionand bursting of the vulva (40% of animals, n=40). Consistent withmir-84:: gfp expression in seam cells, it was found that mir-84(+++)animals also exhibited precocious seam cell terminal differentiation andalae formation in the L4 stage, a characteristic seen in precociousdevelopmental timing mutants. In fact, let-7 over-expressing strainsalso exhibit precocious seam cell terminal differentiation in the L4stage (Reinhart et al., (2000) Nature 403, 901-906). In contrast,animals carrying an array containing a construct identical tomir-84(+++) except for a 75 nucleotide (nt) deletion of sequencesencoding the predicted pre-mir-84 (Δmir-84 (i++)) did not display anyvulval or seam defects, demonstrating that the phenotypes observed inmir-84(+++) are dependent on the miR-84 sequence.

A search was conducted for let-7 family miRNA complementary sequences(LCS) in the 3′UTRs of all genes known to play a role in vulvaldevelopment (Table 2). LCSs have the potential to bind all members ofthe let-7 family, including mir-84. Approximately 11 vulval genescontained at least one LCS (Table 2), raising the possibility that thelet-7 family may regulate multiple genes in the vulva. In this analysisthough, let-60/RAS stood out due to the high number of LCS sites.

TABLE 2 LCSs in the 3′UTRs of Known Vulval Genes Gene Chromosome LCS in3′UTR eor-1 IV Yes had-1 V Yes let-60 IV Yes lin-3 IV Yes lin-9 III Yeslin-11 I Yes lin-36 III Yes lin-39 III Yes lin-45 IV Yes mpk-1 III Yessem-5 X Yes

mir-84 overexpression partially suppresses let-60/RAS gain of functionphenotypes. let-60/RAS is active in P6.p following a lin-3 EGF signalfrom the anchor cell that activates a MAPK signal transduction cascadetransforming P6.p to the 1⁰ vulval fate (Han and Sternberg, (1990) Cell63, 921-931). Since mir-84 is expressed in all VPCs except P6.p, thepossibility that mir-84 negatively regulates expression of let-60/RAS incells not destined to adopt the 1⁰ fate was examined. Activatingmutations in let-60/RAS cause multiple VPCs (including the non-P6.pVPCs) to adopt 1⁰ or 2⁰ fates leading to a multivulva (Muv) phenotype(Han et al., 1990). It was found that over-expression of mir-84partially suppressed the Muv phenotype of let-60(gf) mutations. In thestudy, 41% (n=51) of let-60(ga89) (Eisenmann and Kim, (1997) Genetics146, 553-565) animals displayed a Muv phenotype, while only 13% (n=168)did so when also over-expressing mir-84 from a multi-copy array(p<<0.0001 Chi square test). The same suppression was observed with asecond let-60(gf) allele, let-60(n1046) (Han et al., (1990) Genetics126, 899-913): 77% (n=39) of let-60(n1046) animals displayed a Muvphenotype, while only 50% (n=113) did so when also over-expressingmir-84 (p<<0.0001 Chi square test). let-60(n1046) animals displayed anaverage of 1.54 pseudovulvae per animal compared to an average of 0.66pseudovulvae per let-60(n1046) animal over-expressing mir-84. For bothlet-60(gf) alleles, animals exhibiting low mosaicism for the myo-3:: gfpco-injection marker, were completely suppressed, suggesting that thepartial suppression was likely due to mosaicism of the transgeneicarray. Neither an empty vector control (TOPO) (n=111) (p=0.1435 Chisquare test), nor the Δmir-84 (+++) array (n=129), suppressed the Muvphenotype of let-60(n1046). For all let-60(gf) experiments, threeindependent lines behaved similarly (FIG. 13C).

The let-60RAS 3′UTR confines expression to P6.p. The promoter oflet-60/RAS drives reporter expression in all VPCs (Dent and Han, (1998)Mech Dev 72, 179-182). However, the transgenic reporters used in thisearlier work did not include the let-60 3′UTR. GFP was fused to thelet-60 3′UTR and drove GFP expression in all the VPCs using theVPC-specific lin-31 (Tan et al., (1998) Cell 93, 569-580) promoter(gfp60). In the late L2 and early L3 stages, GFP was expressed in allthe Pn.p cells, but by mid to late L3 stages, GFP was largely restrictedto the P6.p cell (FIG. 6B), with some expression in the P5.p and P7.pcell descendants. A similar fusion construct in which the let-60 3′UTRwas replaced by the unregulated unc-54 3′UTR showed GFP expression inall Pn.p cells (FIG. 6C). Since the lin-31 promoter is active in allPn.p cells (Tan et al., (1998) Cell 93, 569-580), this resultdemonstrates that the let-60/RAS 3′UTR is sufficient to down-regulate areporter gene in the non-P6.p cells.

The let-60 3′UTR was replaced with the unregulated unc-54 3′UTR in alet-60 genomic DNA fragment. While one could generate viable lines usinga let-60:: let-60(+)::let-60 3′UTR construct at 10 ng/μl, it was notpossible to generate viable transformants using this let-60::let-60(+)::unc-54 3′UTR construct, even at 0.1 ng/μl. The resultssuggest that the removal of the let-60 3′UTR may severely over-expresslet-60 and cause lethality.

let-60/RAS is a likely target of mir-84 in the vulva. Previous work hasdemonstrated that VPCs are sensitive to the levels of let-60/RAS (Beitelet al., (1990) Nature 348, 503-509; Han et al.(1990)). Animals carryingextra copies of the wild-type let-60/RAS gene display a Muv phenotype,where non-P6.p VPCs can adopt the 1° fate. The data strongly suggestthat mir-84 negatively regulates let-60 in non-P6.p VPCs. First, mir-84is complementary to multiple sites in the let-60 3′UTR. Second, mir-84is expressed in a reciprocal manner to let-60 in the VPCs. miR-84 islargely absent from P6.p, at the same time as the let-60 3′UTR confinesGFP expression mainly to the P6.p cell lineage. Finally, mir-84over-expression partially suppresses the effects of activating mutationsin the let-60 gene. mir-84 modulates the expression of let-60/RAS innon-P6.p VPCs to reduce flux through the RAS/MAPK signaling pathway andhence decrease the likelihood that these cells will also adopt the 1°fate. However, mir-84 is clearly not the only regulator of let-60/RAS innon-P6.p cells: daf-12(rh61) mutants do not express mir-84 in any VPC(n=60 animals), and yet daf-12(rh61) animals do not display a Muvphenotype. Other known factors, e.g. synmuv genes, (Berset et al.,(2001) Science 291, 1055-1058; Ceol and Horvitz, (004) Dev Cell 6,563-576; Hopper et al., (2000) Mol Cell 6, 65-75; Lee et al., (1994)Genes Dev 8, 60-73; Wang and Sternberg, (2001) Curr Top Dev Biol 51,189-220; Yoo et al., (2004) Science 303, 663-666; Yoon et al., (1995)Science 269, 1102-1105) or unknown factors may also regulate let-60/RASsignaling in these cells.

The combined results provide strong evidence that let-7 and mir-84regulate let-60/RAS expression through its 3′UTR in seam and vulvalcells, cells in which they are all naturally expressed. Given that the3′UTR of let-60/RAS contains multiple let-7/mir-84 complementary sites,it is expected that this regulation is direct.

let-7 complementary sites in human RAS 3′UTRs. Numerous miRNAs arealtered in human cancers (Calin et al., (2002) Proc Natl Acad Sci USA99, 15524-15529; Calin et al., (2004) Proc Natl Acad Sci USA 101,2999-3004; Michael et al., (2003) Mol Cancer Res 1,882-891; Tam et al.,(2002) J Virol 76, 4275-4286) and three of the best understood miRNAs,lin-4 (Lee et al., (1993) Cell 75, 843-854), let-7 (Reinhart et al.,(2000) Nature 403, 901-906) and bantam (Brennecke et al., (2003) Cell113, 25-36), all regulate cell proliferation and differentiation. Theclosest human homologues of let-7 and mir-84 are the H.s. let-7 familymiRNAs (Lagos-Quintana et al., (2002) Curr Biol 12, 735-739; Pasquinelliet al., (2000) Nature 408, 86-89). let-60/RAS (SEQ ID No. 87) is the C.elegans orthologue of human HRAS (SEQ ID No. 84), KRAS (SEQ ID No. 86),and NRAS (SEQ ID No. 85) (FIGS. 13A-B), which are commonly mutated inhuman cancer (Malumbres and Barbacid, (2003) Nat Rev Cancer 3, 459-465),including lung cancer. It was found that all three human RAS 3′UTRscontain multiple putative let-7 complementary sites with features ofvalidated C. elegans LCSs (SEQ ID Nos. 36, 37, 38, 40, 41, 42, 43, 44,45, 46, 55) (FIGS. 4B-D). Many of these are conserved in rodents,amphibians and fish (FIG. 14 and FIG. 15 (SEQ ID Nos. 39, 97, 98, 99,100, 101, 102, 103), suggesting functional relevance. The presence ofputative LCSs in human RAS 3′UTRs indicates that mammalian let-7 familymembers may regulate human RAS in a manner similar to the way let-7 andmir-84 regulate let-60/RAS in C. elegans.

Human RAS Expression is Regulated by-let-7 in Cell Culture.

Microarray analysis performed by Ambion, Inc. on six different celllines revealed that HepG2 cells express let-7 at levels too low todetect by microarray analysis. Therefore, by request, Ambion, Inc.,transfected HepG2 cells with a double-stranded (ds) RNA that mimics thelet-7a precursor. Consistent with the prediction that RAS expression isnegatively regulated by let-7, immunofluorescence with a RAS-specificantibody revealed that the protein is reduced by approximately 70% inHepG2 cells transfected with exogenous let-7a miRNA relative to the samecells transfected with a negative control miRNA (FIG. 7A). The proteinexpression levels of GAPDH and p21^(C1P1) were largely unaffected by thetransfected let-7a and negative control pre-miRNAs (FIG. 16A),indicating that let-7a regulation is specific to RAS. To confirm thatthe RAS antibody is specific to RAS protein in the transfected cells,HepG2 cells were also independently transfected with two exactlycomplementary siRNAs targeting independent regions of NRAS. Both siRNAsreduced cell fluorescence by more than 60% as compared to negativecontrol siRNA-transfected cells (FIG. 16B).

It was predicted that cells expressing native let-7 may express less RASprotein and that inhibition of let-7 may lead to derepression of RASexpression. To test this, by request, Ambion, Inc. transfected HeLacells, which express endogenous let-7 (Lagos-Quintana et al., (2001)Science 294,853-858; Lim et al., (2003) Genes Dev 17, 991-1008), withanti-sense molecules designed to inhibit the activity of let-7(Hutvagner et al., (2004) PLoS Biol 2, E98; Meister et al., (2004) RNA10, 544-550). Reducing the activity of let-7 in HeLa cells resulted inan about 70% increase in RAS protein levels (FIG. 7B). These results,combined with the reciprocal experiment using pre-let-7 miRNAs discussedabove, indicates that let-7 negatively regulates the expression of RASin human cells.

The 3′UTR of human NRAS and KRAS was fused to a luciferase reporter geneand these constructs transfected along with transfection controls intoHeLa cells. NRAS contains two naturally occurring 3′UTRs that utilizealternative polyadenylylation and cleavage sites, such that one of the3′UTRs is 2.5 kb longer than the other. It was found that while the longNRAS 3′UTR strongly repressed reporter expression compared to anunregulated control 3′UTR (FIGS. 8A-B), the short NRAS 3′UTR led to onlyslight, but reproducible, repression of the reporter. The short 3′UTRcontains 4 LCSs, while the long form contains 9 LCSs. The KRAS 3′UTRalso repressed the luciferase reporter (FIG. 8A-B), while HRAS was nottested. The results demonstrate that the 3′UTRs of NRAS and KRAS containregulatory information, sufficient to down-regulate the reporter.

As with the endogenous RAS experiments described above, the reciprocalexperiment was performed wherein HeLa cells were transfected with theRAS 3′ UTR reporter constructs and the let-7a anti-sense inhibitormolecule (or a control scrambled molecule). Cells transfected with thelet-7a inhibitor relieved repression exerted on the reporter relative tothe control transfections (FIG. 8C). Since, a loss in the extent ofdown-regulation is observed when let-7 is inhibited, these resultsstrongly indicate that let-7 regulates NRAS and KRAS in human cellsthrough their 3′UTRs.

let-7, RAS and lung cancer. Like let-60/ras, human RAS isdose-sensitive, since over-expression of RAS results in oncogenictransformation of human cells (McKay et al., (1986) Embo J 5, 2617-2621;Pulciani et al., (1985) Mol Cell Biol 5, 2836-2841). It is plausiblethat loss of miRNA control of RAS could also lead to over-expression ofRAS and contribute to human cancer. Recent work has mapped let-7 familymembers to human chromosomal sites implicated in a variety of cancers(Calin et al., (2004) Proc Natl Acad Sci USA 101, 2999-3004). Inparticular let-7a-2, let-7c and let-7g have been linked to smallchromosomal intervals that are deleted in lung cancers (Calin et al.,(2004) Proc Natl Acad Sci USA 101, 2999-3004), a cancer type in whichRAS mis-regulation is known to be a key oncogenic event (Ahrendt et al.,(2001) Cancer 92, 1525-1530; Johnson et al., (2001) Nature 410,1111-1116).

miRNA microarray analysis was performed by Ambion, Inc. to examineexpression levels of members of the let-7gene family in tissue fromtwenty-one different cancer patients, including twelve lung cancerpatients with squamous cell carcinomas (stage IB or IIA). let-7 ispoorly expressed in lung tumors, as shown by expression of let-7 in 21breast, colon, and lung tumors relative to associated normal adjacenttissue (“NAT”). Fluorescently labeled miRNA was hybridized tomicroarrays that included probes specific to let-7a and let-7c.Fluorescence intensities for the tumor and NAT were normalized by totalfluorescence signal for all elements and the relative average signalfrom the let-7 probes in the tumor and normal adjacent samples areexpressed as log ratios. let-7a and let-7c had similar profilessuggesting cross-hybridization between the two closely related miRNAs.let-7 miRNAs were reduced in expression in a number of the tumorsrelative to the normal adjacent tissue samples from the same patients.let-7 was expressed at lower levels in all of the lung tumor tissues(FIG. 9), but only sporadically in other tumor types. A similar findingwas independently discovered (Takamizawa et al., (2004) Cancer Res 64,3753-3756). On average, let-7 was expressed in lung tumors at less than50% of expression in the associated normal lung samples. Northernanalysis was used to measure let-7c in the tumor and NAT samples for thetwo patients from which RNA was purified (samples represented by thefirst and fifth lung cancer bars in FIG. 9). Consistent with themicroarray results by Ambion, Inc., northern analysis verified that theexpression of let-7c was 65% lower in the tumor of patient #1 and 25%lower in the tumor of patient #5. Seven of eight examined samples alsohad on average 30% less let-7g expression in the tumor tissue (FIG. 17).The miRNA arrays were used to compare the lung tumors and NAT includedprobes for 167 total miRNAs. The expression of the vast majority ofthese were unchanged in the lung tumors indicating that let-7 might beimportant in lung cancer. In theory, down regulation of let-7 couldresult in up-regulation of RAS and thus induce or accentuateoncogenesis.

To test this hypothesis, Ambion, Inc. isolated total RNA and totalprotein from the tumor and normal adjacent tissues of three new lungcancer patients with squamous cell carcinoma. The RNA samples were splitand half was used for northern analysis to measure let-7c and U6 snRNA.The other halves of the RNA samples were used for real-time PCR tomeasure the NRAS mRNA, 18S rRNA, and B-actin mRNA. The protein sampleswere used for western analysis to assess RAS and GAPDH protein levels.As seen in FIG. 9, RAS protein was present in the tumors at levels atleast ten-fold higher than in the normal adjacent samples from the samepatients. Consistent with the miRNA array results of Ambion, Inc. forother lung cancer samples, all three lung tumor samples had 4- to 8-foldlower levels of let-7 than did the corresponding NAT samples. The firstand third lung cancer samples had similar levels of NRAS mRNA in boththe tumor and NAT while the second sample pair had significantly higherlevels of NRAS mRNA in the tumor sample. RAS protein levels correlatepoorly with NRAS mRNA levels but very well with let-7 levels, suggestingthat the expression of the oncogene is significantly influenced at thelevel of translation, consistent with the known mechanism of let-7 ininvertebrates.

The reciprocal expression pattern between let-7 and RAS in cancer cellsclosely resembles what was seen with let-7 and RAS in C. elegans and inthe human tissue culture experiments. The correlation between reducedlet-7 expression and increased RAS protein expression in the lung tumorsamples indicates that one or more members of the let-7 gene familyregulates RAS expression in vivo and that the level of expression of themiRNA is an important factor in limiting or contributing to oncogenesis.

These results demonstrate that the let-7 miRNA family negativelyregulates RAS in two different C. elegans tissues and in two differenthuman cell lines. Strikingly, let-7 is expressed in normal adult lungtissue (Pasquinelli et al., (2000) Nature 408, 86-89), but is poorlyexpressed in lung cancer cell lines and lung cancer tissue (Takamizawaet al., (2004) Cancer Res 64, 3753-3756). The expression of let-7inversely correlates with expression of RAS protein in lung cancertissues, suggesting a possible causal relationship. In addition,over-expression of let-7 inhibited growth of a lung cancer cell line invitro (Takamizawa et al., (2004) Cancer Res 64, 3753-3756), suggesting acausal relationship between let-7 and cell growth in these cells.

These results demonstrate that the expression of the RAS oncogene isregulated by let-7 and that over-expression of let-7 can inhibit tumorcell line growth. The combined observations that let-7 expression isreduced in lung tumors, that several let-7 genes map to genomic regionsthat are often deleted in lung cancer patients, that over-expression oflet-7 can inhibit lung tumor cell line growth, that the expression ofthe RAS oncogene is regulated by let-7, and that RAS is significantlyover-expressed in lung tumor samples strongly implicates let-7 as atumor suppressor in lung tissue and provides evidence of a mechanismforming the basis for treatment.

Example 2 Expression Patterns of let-7 and mir-125, the lin-4 Homologue

It was found that both let-7 and mir-125, the lin-4 homologue, areexpressed in a variety of adult tissues, with prominent expression inthe lung and brain. Interestingly, both are expressed at low levels inthe pancreas and testis. Past work has shown that the C. elegans lin-4and let-7 miRNAs are temporally expressed (Reinhart, B., et al., (2000)403:901-906 and Feinbaum, R. and V. Ambros, (1999) Dev Biol210(1):87-95). Similarly, it has been shown that the mammalianhomologues for these miRNAs are also temporally expressed during mousedevelopment. Northern blots reveal that let-7 and mir-125 have verysimilar expression profiles. Both become expressed at around day E9.5.Interestingly, this coincides approximately with the time of lungorganogenesis, and when other major organs begin to develop.

An in situ protocol using an oligonucleotide based on the mouse sequenceof the let-7c miRNA, which has been digoxigenin-labeled, has beendeveloped. Since it has been confirmed by northern analysis that let-7cis expressed at E12.5, frozen sections taken from similarly aged embryoshave been analyzed. Preliminary results show let-7 is expressed in thelung epithelium by in situ. In addition to other cancers, these resultsindicate that let-7 is useful as a therapeutic for lung cancer therapybecause let-7 is a natural compound in lung cells.

Example 3 Effect of Inhibition and Overexpression of let-7 in LungCancer Cells

Inhibition of let-7 function in A549 lung cancer cells via transfection(with anti-let-7 molecules) causes increased cell division of A549 lungcancer cells (FIG. 18A), while let-7 over-expression (with transfectedpre-let-7) caused a reduction in A549 cell number (FIG. 18B). Theseresults are consistent with the tumor suppressing activity of let-7.Moreover, the let-7 over-expression phenotype resembled that caused byMYC down-regulation (FIG. 18A), suggesting that the effects of let-7 oncell proliferation may also be through repression of MYC. Preliminaryevidence indicates that MYC is also a direct target of let-7 in humancells. Therefore, these results indicate that let-7 may be a potentialtherapeutic in cancers with aberrant expression of MYC.

Example 4 let-7 Affects Expression of MYC and BCL-2

let-7 regulates RAS, MYC and BCL-2 protein levels, all three of whichare major cancer oncogenes. Addition of let-7 to HepG2 cells (that donot make let-7 endogenously, reduces the expression of all three ofthese important oncoproteins (FIGS. 20, 7A, and 8). HeLa cells that makeendogenous let-7 express increased levels of all three oncoproteins whentransfected with an anti-let-7 inhibitor (FIGS. 20A, B and 7B). Theseresults demonstrate that let-7 represses expression of these genes.Multiple mir-125 and mir-143 complementary sites have been identified inthe human KRAS and BCL2 3′UTRs and it is expected these oncomirs mayalso regulate these oncogenes in a manner similar to that seen withlet-7. The results indicate that let-7 is a master regulator of cancerpathways, regulating proliferation (RAS and MYC) and survival pathways(BCL2). It is possible that let-7 also regulates telomerase (TERT) andangiogenesis (VEGF) pathways (Table 1). Since cancer is the result ofmultiple genetic mutations, these results indicate that introduction oflet-7 to cancer patients could repress the expression of multipleoncogenes, and provide an effective therapy (effectively a one drugcocktail).

It is understood that the disclosed invention is not limited to theparticular methodology, protocols, and reagents described as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims. Those skilled in the art willrecognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. Such equivalents are intended to beencompassed by the following claims.

1. A method for inhibiting the expression of an oncogene containing alet-7 complementary site (LCS) selected from the group consisting ofNRAS, KRAS, HRAS, MYC, MYCL1, MYCN, BCL2, BCL2L1, BCL2L2, TERT, VEGF,EGF, EGFR, ERBB3, GRB2, RAF1, ARAF, MAP2K2, MAPK1, MAPK3, MET, KIT,TP73L(AIX), CCND1, CDK4, MDM2, FES, FURIN, INSL3, CSFIR, MYBL2, MYB,PIK3CD, PIK3C2B, PIK3CG, PIK3R5, AKT1, HLIN-41, VDR, PXR, FOXA1, FOXA2,ASH1L, ARID1B, GR, GLI2,14-3-3zeta, MO25, SMG1, FRAP1, PER2 and AKT3comprising providing an effective amount of a composition comprising alet-7 miRNA and a suitable pharmaceutical carrier to bind to and inhibitexpression of an mRNA encoded by the oncogene or to increase expressionof a let-7 miRNA that binds to and inhibits expression of an mRNAencoded by the oncogene, in a cell of an organism.
 2. The method ofclaim 1 wherein the let-7 miRNA is a mature miRNA.
 3. The method ofclaim 2 wherein the let-7 miRNA is encoded by a nucleic acid.
 4. Themethod of claim 3 wherein the nucleic acid is located on a vector. 5.The method of claim 4 wherein the vector is selected from the groupconsisting of a plasmid, cosmid, phagemid, virus, and other vehiclesderived from viral or bacterial sources.
 6. The method of claim 4wherein the vector further comprises one or more in vivo expressionelements.
 7. The method of claim 6 wherein the in vivo expressionelement is selected from the group consisting of a promoter, enhancer,and combinations thereof.
 8. The method of claim 1 wherein the let-7miRNA is from 21 nucleotides to 170 nucleotides in length.
 9. The methodof claim 8 wherein the let-7 miRNA is from 21 to 25 nucleotides inlength.
 10. The method of claim 1 wherein the let-7 miRNA isadministered to, or expression is increased in the cells of, a patientfor treatment or prevention of cancer.
 11. The method of claim 10wherein the cancer is selected from the group consisting of lung cancer,pancreatic cancer, skin cancer, hematological neoplasms, breast cancer,brain cancer, colon cancer, follicular lymphoma, bladder cancer,cervical cancer, endometrial cancer, esophageal cancer, gastric cancer,head and neck cancer, multiple myeloma, liver cancer, lymphomas, oralcancer, osteosarcomas, ovarian cancer, prostate cancer, testicularcancer, and thyroid cancer.
 12. The method of claim 10 wherein thepatient is undergoing one or more cancer therapies selected from thegroup consisting of surgery, chemotherapy, radiotherapy, thermotherapy,immunotherapy, hormone therapy and laser therapy.
 13. The method ofclaim 1 wherein the organism is a human.
 14. A method for determiningthe sensitivity of a cancer to a let-7 miRNA comprising providing aneffective amount of a composition comprising a let-7 miRNA and asuitable pharmaceutical carrier to bind to an mRNA encoded by anoncogene containing a let-7 complementary site (LCS) selected from thegroup consisting of NRAS, KRAS, HRAS, MYC, MYCL1, MYCN, BCL2, BCL2L1,BCL2L2, TERT, VEGF, EGF, EGFR, ERBB3, GRB2, RAF1, ARAF, MAP2K2, MAPK1,MAPK3, MET, KIT, TP73L(AIX), CCND1, CDK4, MDM2, FES, FURIN, INSL3,CSFIR, MYBL2, MYB, PIK3CD, PIK3C2B, PIK3CG, PIK3R5, AKT1, HLIN-41, VDR,PXR, FOXA1, FOXA2, ASH1L, ARID1B, GR, GLI2,14-3-3zeta, MO25, SMG1,FRAP1, PER2 and AKT3 or to increase expression of a let-7 miRNA thatbinds to an mRNA encoded by the oncogene in a cancerous or transformedcell or an organism with a cancerous or transformed cell; anddetermining if the cancerous or transformed cell growth or viability isinhibited or if expression of the oncogene is inhibited.
 15. The methodof claim 1 or 14, wherein the suitable pharmaceutical carrier is avirus, a liposome, or a polymer.